Bioimaging Instructor

Bioimaging Instructor kdorfman Tue, 06/16/2009 - 18:07

Here are the preparation procedures for Bioimaging labs.

Bioimaging F 2018

Bioimaging F 2018 kdorfman Tue, 07/31/2012 - 15:51

Cells for lab

day date Lab cells stained for
W 9/5 1. intro fixed LL phalloidin or MT, DAPI
F 9/7 2. Image formation fixed LL phalloidin or MT, DAPI
W 9/12 3. making figures none
F 9/14 4. NA fixed LL MT, phalloidin, DAPI
W 9/19 5. Resolution fixed LL MT, DAPI
F 9/21 6. fluorescence fixed LL MT, phalloidin, DAPI
REALLY BRIGHT for photobleaching!
W 9/26 lab practical none
F 9/28 presentation none
W 10/3 7. immunoflluor fixed LL ,
3t3 (fixed?),
live LL on coverslips
unstained
F 10/5 8. organelles fixed LL Golgi,
gamma tub,
Hec 1,
Lap 2,
Alpha actinin,
ZO 1
F 10/12 9. Live cells

0: Pre-sem prep

0: Pre-sem prep kdorfman Wed, 12/07/2011 - 15:49

Pre-semester prep

Check out 20 minute secondary antibodies from Invitrogen

Get 2 boxes each: 20 mm and 14 mm coverslip dishes from Invitro-Scientific, much cheaper than MatTek.

http://www.invitrosci.com/product_detail.php?product_id=21

http://www.invitrosci.com/product_detail.php?product_id=25


Nucleofector kit: Ingenio Electroporation kit, MIR50112

www.mirusbio.com

Fisher: MIR50112 $230.17. More expensive than mirusbio, but mirus charges $35 for shipping.

Solutions

Solutions kdorfman Tue, 12/27/2011 - 19:21

Solutions to have at the beginning of the semester

Amount conc solution
100 mL 10% Tween in water
50 mL 10% Tween in PBS
100 mL 10% Triton-X in water
100 mL 10% Triton-X in PBS
5 L 10X PBS
1 L 1x PBS in 10 mL sterile aliquots
4 L 1x PBS-Tw-Az
25 mL 10% Sodium azide
250 mL 10X HBS
100 mL 10 X Ca-free HBS
500 mL 1x Non-CO2 medium with serum
1000 mL 1x Non-CO2 serum free medium
2 L 1x F10-Hams
250 mL 1x serum free F-10 Hams
1 L 1x DMEM
20 mL DAPI Fluoromount Fisher OB010020 2 bottles to share.

Aliquots

Aliquots kdorfman Tue, 12/27/2011 - 19:27

Students use lots of these:

  • 10 mL PBS
  • 10 mL serum-free-non-CO2 medium
  • 10 mL HBSS
  • 10 mL HBS

Slides

Slides kdorfman Tue, 12/27/2011 - 20:56

slides 2012

slides 2012 kdorfman Fri, 08/23/2013 - 14:07

CHECK THIS

LLCPk-1

coverslips, fixed in 3.5% formaldehyde and stored in PBS-tween-azide in coplin jars in the refrigerator:

  • 30 unmounted,

  • 10 mounted, stained only with DAPI

  • 20 stained for tubulin, and mounted with DAPI

  • 20 stained for actin and tubulin, and mounted with DAPI

  • 10 serum starved, stained for tubulin, mounted with DAPI

    • for Lab 4.1
    • Grow first in regular medium (otherwise they won't stick)
    • Replace medium with serum free medium.
    • Fix after 24 hours
  • 10 Arrested with 100nM nocodazole (8 - 16 hours)

    • for Lab 4.1
    • Treat with nocodazole late in the day
    • Fix the next day in para-glut or formaldehyde
    • Stain for tubulin, mount with DAPI

Nocodazole stock is 33 mM in DMSO
Make 100 µL 1 mM: 3 µL (33mM) + 97 µL medium
Make 1 mL 6µM: 6 µL (1mM) + 994 µL medium
Add 1 drop (~50 µL) 6µM to each dish of ~3 mL

OR

Make 30 mL 100 nM: 3 µL (1mM) in 30 mL medium
Change the medium in each dish.

3t3

12 for Lab 3.3, unmounted

slides 2013

slides 2013 kdorfman Fri, 08/23/2013 - 14:08
Lab date # DAPI + fix
1.1 & 1.3 9/4 & 11 10 paraglut
1.2 9/9 10 alpha tub paraglut
2.1 & 2.2 9/16 10 alpha tub, actin paraglut
3.1 9/23 10 alpha tub, actin paraglut
3.2 9/25 2 Golgi formaldehyde (no glut)
2 gamma tubulin paraglut or methanol
2 actin paraglut
2 alpha actinin formaldehyde (no glut)
2 ZO1 methanol or paraformaldehyde
2 LAP2 methanol
2 vinculin formaldehyde

slides 2015

slides 2015 kdorfman Tue, 07/14/2015 - 14:58

LL's for student slides

Fix 90 in formaldehyde; 10 in methanol

Plate 102 in 17 6-well plates.

Lab # coverslips fix stain secondary
1.1 10 form or paraglut
1.2 10 "
1.3 10 "
2.1 10 " anti-tubulin, phalloidin goat anti-rat
2.2 10 " anti-tubulin goat anti-rat
3.1 10 " anti-tubulin, phalloidin FRESH goat anti-rat
3.2 3 form alpha actinin anti-mouse IgM
3.2 3 form anti Golgi anti-mouse IgG
3.2 3 form vinculin
3.2 3 MeOH anti gamma-tubulin
3.2 3 " anti-Hec1
3.2 3 " LAP2
3.3 20 form or paraglut un-mounted for students to stain

organelle slides

organelle slides kdorfman Tue, 07/14/2015 - 14:59

Alpha actinin

Alpha actinin kdorfman Tue, 07/14/2015 - 15:02

Stains adhesions and stress fibers

  • Monoclonal, BM 75.2
  • Fix in Formaldehyde, no glut or meOH
  • 1:200
  • IgM, use mouse FITC secondary that recognizes IgMs

Gamma tubulin

Gamma tubulin kdorfman Tue, 07/14/2015 - 15:03

Stains Centrosomes

  • Monoclonal, GTU-88, Sigma,
  • Fixation: methanol; para/glut
  • Dilution 1:100

Golgi 58K

Golgi 58K kdorfman Tue, 07/14/2015 - 15:04
  • Sigma G2404, mouse monoclonal
  • Formaldehyde (no glut)
  • 1:100

Hec1

Hec1 kdorfman Tue, 07/14/2015 - 15:05

Stains kinetochores

  • Novus biological, recombinant human HEc1, monoclonal
  • Fix in methanol
  • 1:200

LAP2

LAP2 kdorfman Tue, 07/14/2015 - 15:07

Stains nuclear envelope

  • Methanol
  • 1:100
  • BD transduction
  • Mouse IgG

Vinculin

Vinculin kdorfman Tue, 07/14/2015 - 15:08

Focal adhesion plaques

  • Monoclonal, sigma V4505
  • Formaldehyde
  • 1:100

2016 schedule

2016 schedule kdorfman Wed, 09/21/2016 - 20:06

Lab 1 1 DAPI slide per pair

Lab 2

  • same DAPI slide as Lab 1

  • 1 H&E slide from histology

Lab 3 Numerical aperture . same DAPI slide

Lab 4 Resolution.

  • 1 slide newly stained phalloidin actin. Must be bright

  • 1 slide fluorescent beads(PS-Speck Microscope Point Source Kit P7220 from Molecular Probes)

Lab 5 Fluorescence. 1 new, bright 3-color stained slide (DAPI, Phalloidin-actin, FITC microtubules)

Lab 6 Dry lab

Lab 7 Labeling Cells 9/28/16

  • 3 fixed LL coverslips

  • 1 3t3 fixed coverslip

  • 1 live LL coverslip for students to fix

  • paraglut

  • forceps, mounting medium, pasteur pipettes

Lab 8 - Organelles 10/3/16

  • Golgi - anti-58K, mouse, formaldehyde fixation

  • Kinetochore - anti Hec1 - MeOH fixation 1:200 mouse

  • ER anti-calnexin, paraformaldehyde fixation 1:200

  • centrosome - anti gamma-tubulin, paraglut, mouse 1:100

  • nuclear envelope anti LAP2 - MeOH fixation 1:100 mouse

  • lysosomes

  • alpha actinin - mouse 1:200 formaldehyde fixation

Lab 8 Practical (and pipet practice)- 10/5

Lab 9 - Imaging Live Cells 10/11 TUESDAY Live cells on mattek

unknown GFP tagged proteins

Thaw GFP lines Wednesday
Split Thursday (~1/4)
Plate Sunday on Mateks for Tuesday

  • Thaw
    • GFP tubulin
    • actin thaw
    • EB1 - thaw
  • Nucleofect: EGFP-H2B
    • split LL parentals on Sunday, HEAVY
    • nucleofect & plate Monday by noon

Lab 10 10/12

  • Single cells to whole organisms

    • order protists

    • induce GFP E. coli

  • CRISPR: Transform guide RNA plasmid into bacteria

Lab 11.1 Motility I 10/17

  • thaw B16, 3t3 Thursday 10/13/16 (late in the day)
  • plate 9 MatTeks each Fri 10/14/16:
    • 3t3
    • actin GFP LL
    • B16

Lab 11.2 Motility II 10/19

Lab 11.3 Motility III 10/24

Motility presentations 10/26

CRISPR: prepare Cas9/guide RNA plasmid DNA 10/31

CRISPR: prepare repair DNA cassette (PCR, clean up) 11/2

CRISPR: Add Cas9 plasmid + repair DNA to cells 11/7

Lab 13.1 - Cell Signaling 11/9

Lab 13.2 - Cell Signaling 2 11/14

11/16 Wed = Friday schedule

Discussion of independent projects 11/28

Projects 11/30 - 12/14

Final exam period: Final project presentation

2018 cell schedule

2018 cell schedule kdorfman Wed, 12/20/2017 - 18:48
Lab date number cell line live or fixed in stained with notes
1&2 1/23 10 LL parental fixed in paraglut phalloidin learn about scope
3&4 1/30 10 LL parental fixed in paraglut tubulin, phalloidin, DAPI NA
5 2/6 10 LL parental (dense) fixed in paraglut tubulin, phalloidin, DAPI photobleaching
6 2/8 none Image J, photoshop
7 2/13 30 LL parental fixed in paraglut unstained immunofluorescence
10 3t3 fixed in paraglut unstained
10 LL parental live on coverslip to fix and stain
8 2/15 Lab practical
9 2/20 3 LL parental fixed in MeOH gamma tubulin 1:100 (mouse) (C) organelle ID lab
3 LL parental fixed in MeOH Hec1 1:200 (mouse) (didn't do)
3 LL parental fixed in MeOH LAP2 1:200 (mouse) (B)
3 LL parental formaldehyde only Alpha actinin (mouse IgM) 1:200 (D)
3 LL parental formaldehyde only Golgi 58K 1:100 (mouse) (A)
3 LL parental doesn't matter secondary Ab only
9 2/22 10 LL a-tub GFP/RFP myosin live in dish imaging live cells
18 LL live in dish for mitotracker or ceramide
10 LL H2B m cherry live in dish transfected unknowns
10 2/27 GFP E. coli, motile E. coli, Volvox Carolina 152655, Tetrahymena Carolina 131620, Dictyostelium Carolina 55996, Paramecium caudatum Carolina 131554, Chaos (Pelomyxa) carolinensis Carolina 131324
11 3/1 10 3t3 live in dish motility
10 LL parental live in dish
10 melanoma live in dish
10 LL GFP-actin live in dish
12 3/6 9 3t3 live in dish Motility II
12 LL GFP-tub
3 LL GFP actin
5 B16
13 3/8 Posters
14 3/20 26 3t3 live on coverslip Endocytosis I
3/22 18 3T3 live on coverslip Endocytosis II
10 LL parental live on coverslip
18 LL a-tub-GFP live on coverslip
13 3/27 28 3t3 live in dish (dense) Signalling 1
3/29 28 3t3 live in dish (dense) Signalling 2
14 4/3 10 LL-a-tub-GFP live in dish mitosis, cytokinesis
18 LL myo tub live in dish
15 4/5 10 LL- parental live on coverslip for fixation
10 LL GFP-tub (or tub & myo) live in dish for treatment, observation

projects 2018

projects 2018 kdorfman Thu, 04/05/2018 - 22:21
date scope cells reagents
4/10 1 3 LL parentals (coverslip or dish?) ?
2 Volvox Volvox medium (ordered)
3 LL parentals, dense ?
4 4 3t3 live in dish ?
5 4 LL GFP tubulin live in dish ?
6 3 LL GFP tubulin live in dish? ?
7 1 live ll parentals in dish ceramide
8 3 GFP actin (in dish?) cytochalasin D
date scope cells reagents
4/12 1 3 LL parental in dishes
2 Volvox aureus (ordered)
3 dense LL's (reuse Tuesday's cells? could use GFP tubulin
4 6 dishes 3t3 densely plated
5 3 live GFP actin LL's, 1 GFP tubulin LL cytochalasin D, latrunculin
6 3 GFP tubulin in dish
7 2 parental LL in dish ceramide
8 3 GFP tubulin in dishes
date scope cells reagents
4/19 1 5 dishes LL parentals, dense
2 Volvox aureus (will check Monday; order more if needed)
3 will tell us by Tuesday
4 5 3t3 in dishes
5 3 actin GFP, 3 GFP tubulin LLs (dishes)
6 4 GFP tubulin LL's (dishes)
7 2 LL parental in dish NBD ceramide
2 GFP-tubulin coverslip anti-Golgi
8 2 GFP tubulin, 1 GFP actin LLs in dishes
date scope cells reagents
4/24 1 3 LL parentals in dish
2 Volvox
3 4 dense dishes LL parentals
4 3 live 3t3, 3 live LL
5 3 live GFP tub LL, 3 actin
6 4 GFP tubulin LL live
7 3 live LL parentals ceramide
8 2 dishes LL GFP tubulin

2019 F

2019 F kdorfman Thu, 09/12/2019 - 15:46
date prep
9/19 triple stained LLCPk slides: DAPI, rat anti-alpha tubulin & anti-rat-FITC, red phalloidin
9/26 thaw LL flask
9/27 change medium
9/28 split into large flask
9/30 put 5 x 10^5 cells into each of 9 flasks
10/1 students nucleofect
10/3 Start G418 selection of transfected LLs
10/9 Thaw 3t3 into DMEM, spin down, resuspend, plate on coverslips. No adherence (or all dead?) Make new DMEM, replace medium in flasks
10/10 Stop selection - cells look bad. Replace selection medium with regular F10Hams
10/11 very few adherent cells.
10/15 Thaw LL-GFP-alphas,HeLa-H2B-GFP, Mcherry alpha tubulin
(get HeLa GFP alpha tubulin from Pat)
3t3s look fine
split transfected cells into one 25 cm2 flask to grow up, one 12.5 cm2 flask to continue selection
10/16 Plate in coverslip dishes for lab on 10/17 (had to be done in Pat's lab)
10/17 live cells for mitosis (made in Pat's lab: HeLa-GFP-tubulin, LLCPk-GFP tubulin)
thaw LLCPk-GFP-actin for use in motility lab 10/22
thaw LLCPk-GFP-tubulin to test freeze/thaw protocol
split 3t3s to grow up for live cell observations 10/22
split LL parentals in case anyone wants to repeat nucleofection
split nucleofected cells for students; they take over care of them 10/24
10/21 plate (coverslip bottom dishes) 3t3, LL-GFP-actin from flasks
make big flask of LL-GFP-actin to freeze
thaw b16, spin down, resuspend in DMEM, plate (coverslip dishes)
froze 3 vials HeLa-GFP-H2b/mcherry-tubulin
10/22 Bioimaging students split their 2 flasks of nucleofected cells (G418/unselected); made 2 new flasks, 2 coverslip dishes.
10/23 Set up sterile hoods with all equipment students will need.
Freeze 3 vials of 3t3s
10/24 students split nucleofected cells
10/25 split LL-GFP-tub, LL-GFP-actin and freeze 2 vials each
split HeLa-GFP-tub: very strange - some cells never balled up.
10/28 split LL-GFP-tub: 1 flask with 500,000 for nucleofection 10/29, one to keep culture going
split better looking parentals just to keep going
HeLas look OK, but sparse
10/29 students split cells, work on projects
10/30 make 1 flask of LL parentals to nucleofect, 1 flask of LL-GFP-tubulin (plus 1 flask each to perpetuate)
10/31 students work on their cells
11/01 inner incubator door was open, temp was 28C, CO2 was 3
replace medium on HeLas (cultures are pretty sparse)
replace medium on parentals
split LL-GFP-actin to 12.5 flask
11/04 plate 2 coverslip dishes of parental LL's for ER Tracker treatment
make 2 12.5 flasks for propagation
11/05
11/06 split LL-GFP-tubulin: 2 full flasks for nucleofection, one for propagation
aliquot 40 mL trypsin
freeze 3 vials HeLa-GFP-tubulin
one flask HeLa-GFP-tubulin in case students want it
split flask of LL-GFP-actin in case students want it
11/08 Kate sick
11/10 split parentals
split LL-GFP-actin
refeed HeLa-GFP-tubulin
aliquot new F10-Hams & DMEM
11/11 Split BL & AM nucleofected cells: 1 flask for nucleofection, one for propagation
G418 selection @ 2 mg/mL
11/12 students work on cultures
11/13 plate LL-GFP tubulin for nucleofection on 11/14 (with students)
11/14 pat brings over IF LL's (intermediate filament eGFP vimentin)
11/15 check IF flasks: all light. Split on Monday
split LLCPK parentals
split HeLa-GFP-tubulin
11/18 8:30 am help students (AZ) count to prep cells (LL-IF-GFP) for nucleofection
split IF flasks for freezing (2 75cm2 flasks), student projects (2 25 cm2 flasks- each with 500,000 cells in case anyone wants to nucleofect them on Tuesday)
11/19 split LL-GFP-tubulin: 1 heavy, 1 light
11/20 split heavy LL-GFP-tubulin, count, put 500,000 in flask for Sam & Brian to nucleofect
11/21 students work
11/22 LLCPK GFP-IF: freeze 3 (2?) vials from T75 flask; split into T25 flask "in case"
LLCPK GFP-tubulin: split light
J & G: split; will split again 11/29 to make a nucleofection flask 12/1
LLCPK Lifact Actin 488 split into T75 flask for freezing; small flask "in case"
KTRM: change medium in dish and flask
LLCPK parentals: split lightly "in case"
HeLa GFP-Tubulin split lightly "in case"
11/25 ASBK split 2 flasks
KTRM:
GOJC H2B: split
Z&A: actin split 1 flask + G418,
Z&A IF change medium plut G418
SLBM split GFP-Tub 2
AMBL IF+Actin split plus G418 reduced by half
SACW will come in to do their own
11/27 Freeze LifAct-488 LL
SLBM split "s1" (or wait till 11/29?)
SLBM split s2 flask LL-red-mitochondria to a 75 flask to freeze Friday
11/29 LLGFP-GFP-tub split (so students can split on 12/1 for nucleofection 12/2)
SLBM freeze T75 (get labels from Sam & Brian)
HeLas look OK, but sparse

Frozen cell lines

2021

2021 kdorfman Mon, 07/26/2021 - 16:51

Class Schedule:

date day cells reagents
9/2 Th fixed HeLa H2B-GFP mcherry-alpha Tubulin 10 untreated, and 24 treated with VPA, nocodazole, trichostatin A, cytochalasin D, latrunculin
9/7 Tu
9/9 Th
9/14 Tu PS-Speck slides B&G
9/16 Th
9/21 Tu photo bleaching HeLa slides
9/23 Th B16 on coverslips Para-glut
Rat anti-tubulin
goat anti-rat
PBS-Tw-Az
9/28 Tu 1 flask B16/pair passaging
9/30 Th view dish from previous lab
split for nucleofection
mini-prep EB1
10/5 Tu
10/7 Th
10/12 Tu
10/14 Th
10/19 Tu
10/21 Th
10/26 Tu Students split cultures from Drew into 4-chambered dishes
10/28 Th
11/2 Tu Students split onto 4 coverslips, 1 flask/pair 6-well dishes, sterile coverslips
11/4 Th view NLS-GFP cells from Drew's lab, IF PFA
rabbit anti 5mC
rabbit anti-DNMT1
goat anti-rabbit green (try red next time)
11/9 Tu
11/11 Th Veteran's Day
11/16 Tu
11/18 Th
11/23 Tu Th schedule
11/25 Th Thanksgiving
11/30 Tu
12/2 Th
12/7 Tu

Cell passaging

Cell passaging kdorfman Wed, 09/29/2021 - 17:07

Students split a flask of B16s into a new flask and a coverslip-bottom dish for viewing next class

  • 1 flask of B16-30 (wild type) per pair (at least 50% confluent)

  • 1 flask, 1 coverslip-bottom dish per student

  • 1 50 mL tube of DMEM per pair

  • 1 15 mL tube of PBS per pair

  • 1 0.5 mL aliquot of trypsin per pair

  • 5 mL serologicals

  • sterile transfer pipettes

  • 70% ethanol spray bottles

  • lots of gloves

  • handout

Lab 1 prep

Lab 1 prep kdorfman Wed, 08/25/2021 - 16:09
no. treated with conc [stock] time
10 untreated
2 VPA (valproic acid) 1 4 uM 20 uM over night
4 nocodazole 2 100 nM 1 mM (make 1uM 1st) overnight
2 trichostatin A 3 200 nM overnight
4 Cytochalasin D 4 250nM 10 mM 1 hour
2 Latrunculin 5 1 uM 1 mM 1 hour (in serum free medium!)

  1. Get VPA from Drew. 5X final conc.
    Add 0.5625 mL to each well containing 2.25 mL medium. ↩︎

  2. Add 97 uL DMSO to the 3 uL in tube to make 100 uL 1mM.
    Make 2mL 1uM in medium (2 uL 1mM + 1.998 mL medium).
    Add 0.25 mL to the 2.25 mL in each well. ↩︎

  3. TSA 10 mM from freezer.
    Make 1 mL 10 uM (1 uL TSA + 999 uL medium).
    Add 45 uL to each well. ↩︎

  4. Add 90 uL DMSO to the tube(makes 1 mM)
    Dilute this 1:1000 into medium (makes 1uM) (5X)
    Add .256 mL to each well ↩︎

  5. Mix 5 uL into 5 mL serum free medium (can use Fluorobrite)
    Replace the medium in each well with 2.5 mL of Latrunculin-medium mix ↩︎

2022 (Stephens)

2022 (Stephens) kdorfman Fri, 06/03/2022 - 18:20

Pre-semester prep:

6 slides of each cell line (B16 30, 31, 32) all with TRITC phalloidin & DAPI

3 each:

day date lab prep
Th 9/29 image IF slides from previous lab cell culture demo (9 flasks for students, 18 coverslip bottom dishes for 477H, 8 for Kari
Tu 10/4 live cell observations w/ NucBlue & Mitotracker
split into 1 25 cm2 flask for nucleofection
Eb in liquid culture
Th 10/6 split again, miniprep
Tu 10/11 nucleofection aliquot nucleofection reagents
nucleofection into dish, also flask?
Th 10/13 live cell imaging of transfected cells
Tu 10/18 midterm
Th 10/20 Plan CURE projects
Tu 10/25 IF on coverslips: 24 B16 30, 12 B16 31, 12 B16 32 plate on M 10/24
Lamin Ac (mouse) +alpha tubulin (rat), goat anti rat red, goat anti mouse green fix in paraformaldehyde
Hec1 (mouse) + alpha tubulin (rat), goat anti rat red, goat anti mouse green humid chambers, colored markers, DAPI, slides

Cell culture

Cell culture kdorfman Mon, 07/09/2012 - 19:38

Tissue Culture Facility

Tissue Culture Facility margaret Thu, 10/27/2011 - 20:27

Hoods When beginning work in a hood, spray all walls and the working surface with 70% ethanol and wipe clean.

When you have finished working in a hood: please leave it empty, spray the walls and working surface with 70% ethanol and wipe, and make sure that the gas is off. Leave the fan on and turn on the UV light.

Incubators Immediately remove any contaminated dish or flask from the incubator and treat itwith 30% bleach. Wipe the shelf it was on with 70% ethanol. Notify any individuals with cells in the incubator of the contamination.

If you are using an incubator that requires CO2: Always check the water level in the bottom of the incubator, if level is low add water. You are responsible for checking the level of CO2 in the tanks, if pressure falls below 800 psi the tank is emptying. After having received instructions, connect a full tank to the incubator and order a replacement (or notify someone).

Microscopes Please make sure the microscopes are clean and covered when not in use.

Pipets Dispose of all glass Pasteur pipets in the glass only bin. Disposable plastic pipets should be placed in trash (in the plastic sleeve). Reusable pipets should be placed tip up in the pipet jar.

Supplies Label all items in the refrigerator with your name and date. Items that not labeled will be discarded.

Vacuum Pump When you are done, please empty and rinse flask with 30% bleach.

Other Keep the area clean at all times. Dishes and flasks of cells to be discarded should be treated with 30% bleach before trashing.

Bulk coverslip staining

Bulk coverslip staining kdorfman Wed, 09/08/2021 - 20:03
  • Grow cells on coverslips in 6-well plates

  • Remove coverslips using a fine-tipped forceps when desired cell density is reached, and put in coverslip rack, cell side facing "cells" label on holder.

![](coverslip rack.jpg)

  • Put holder in 100 mL beaker containing ~60 mL PBS with a tiny stir bar. Rinse by stirring for ~5 minutes.

  • Transfer holder to a second 100 mL beaker containing ~60 mL of fixative. Fix with stirring for ~10 minutes

  • Transfer holder to a 3rd beaker containing PBS-Tw-Az. Rinse by stirring for ~5 minutes.

  • Repeat with another beaker of PBS-Tw-Az.

  • Stain or mount coverslips right away, or store them in the holder in a brown jar with fresh PBS-Tw-Az. They may last up to a month in the refrigerator. Longer than that, they will start to fall off the coverslip.

CO2 Incubator

CO2 Incubator kdorfman Mon, 07/09/2012 - 19:41
  • 37C
  • 5% CO2
  • pan of water in bottom
  • Thermo Scientific Isotemp Gas Line Filter:

    • Fisher 15-497-026 (Thermo Scientific 760210) $176.54/Pack of 10
  • Thermo Scientific Replacement HEPA Main Filter:

    • Fisher 15-497-022 (Thermo Scientific 760175) $60.60 each
    • Fisher 15-497-023 (Thermo Scientific 760209) $308.62/Pack of 4
  • Manuals under incubator in 371

Counting cells

Counting cells kdorfman Thu, 09/12/2019 - 16:58

Just before plating, after cold medium has been added to the trypsinized cells,

  • mix 0.1 mL cell suspension with 0.1 mL 0.4% trypan blue. Live cells will not take up trypan blue. (Use a different dilution factor if cells are uncountable)

  • inject 10 uL to one side of the hemocytometer (see image attached below).

  • Inspect at 10x.

  • Count the number of live (clear) cells in all 9 squares of the hemocyometer. (Volume = 3mm * 3 mm * 0.1 mm = 0.9 uL) (Count in 4 1mm x 1mm squares if cells are very numerous)

  • Calculate the live cell concentration (# live cells * 1.11)/1uL for 9 squares,
    (# live cells * 2.5/uL for 4 squares)

  • correct for dilution factor

If you count live (clear) cells
in 1 mm x 1 mm squares
with a dilution factor of dilution factor
there are live cells per uL
to get live cells,
you need uL

Hemocytometer Cautions

Hemocytometer Cautions kdorfman Fri, 05/29/2020 - 13:11

Use of the Hemacytometer for the Determination of Cell Numbers

Counting cells by the use of a hemacytometer is a convenient and practical method of determining cell numbers in the case that the Coulter counter is out-of-order temporarily. (It is not that bad.) The hemacytometer consists of two chambers, each of which is divided into nine 1.0 mm squares. A cover glass is supported 0.1 mm over these squares so that the total volume over each square is 1.0 mm x 0.1 mm or 0.1 mm3, or 10-4 cm3. Since 1 cm3 is approximately equivalent to 1 ml, the cell concentration per ml will be the average count per square x 104.

Hemacytometer counts are subject to the following sources of error:

  • 1. Unequal cell distribution in the sample
  • 2. Improper filling of chambers (too much or too little)
  • 3. Failure to adopt a convention for counting cells in contact with the boundaries lines or with each other (be consistent)
  • 4. Statistical error

With careful attention to detail, the overall error can be reduced to about 15%. It is assumed that the total volume in the chamber represents a random sample. This will not be a valid assumption unless the suspension consists of individual well-separated cells. Cell distribution in the hemacytometer chamber depends on the particle number, not particle mass. Thus, cell clumps will distribute in the same way as single cells and can distort the result. Unless 90% or more of the cells are free from contact with other cells, the count should be repeated with a new sample. A sample will not be representative if the cells are allowed to settle before a sample is taken. Always mix the cell suspension thoroughly before sampling. The cell suspension should be diluted so that each such square has between 20 - 50 cells (2-5 x 10 5 cells/ml). A total of 300 - 400 cells should be counted, since the counting error is approximated by the square root of the total count. A common convention is to count cells that touch the middle lines (of the triple lines) to the left and top of the square, but do not count cells similarly located to the right and bottom. Hemacytometer counts do not distinguish between living and dead cells. A number of stains are useful to make this distinction. Trypan blue among others (Erythrosin B, Nigrosin) can be used: the nuclei of damaged or dead cells take up the stain. If more than 20% of the nuclei are stained, the result is probably significant. Although the trypan stain distinction has been questioned, it is simple and gives a good approximation. See procedure here

References:

  • 1. Berkson, J., T. B. Magath and M. Hurn (1939). Am. J. Physiol. 128, 309.
  • 2. Sanford. K.K., W.R. Earle, V.J. Evans, H.K. Waltz and J.E. Shannon (1951).
  • 3. Absher, M. in Tissue Culture Methods and Applications, Eds. Kruse, P.F. and Patterson, M.K., Jr. Academic Press, N.Y., 1973, p.395.
  • From the Laboratory of Dr. Allan Bradley

    Baylor College of Medicine, Houston, Texas

    Culturing S-cells

    Culturing S-cells kdorfman Fri, 05/29/2020 - 15:44

    Cells are in suspension -- they are not dead!

    To make conditioned medium:

    • spin cells to bottom of conical tube (be gentle!!).
    • Save the supernatant in a labeled tube that has the date and is labeled “conditioned medium”.
    • Store extra in fridge.

    To grow cells:

    • Grow cells at room temperature, no CO2.
    • Passage every 3-4 days. Dilute cells 1:5 each split and use 20% conditioned medium.

    • Put cells from flask in tube; spin; take off and save supernatant (this is your conditioned medium!)

    • Resuspend pellet (gently) in 5 mL medium.
    • Add to clean flask
      • 1 mL conditioned medium (supernatant)
      • 1 mL cell suspension
      • 3 mL fresh medium
    • To induce gene expression (e.g, tubulin) add copper sulfate to the medium

    To make medium for growth of Drosphila cells:

    Schneider's medium + 10% heat-inactivated FCS.

    Freezing S2 cells

    Spin down a 3-4 day old flask.

    Resuspend in 4 mL of S2 medium with serum and antibiotic.

    Add 1mL DMSO (20% final).

    Make 1mL aliquots.

    Put in -80 in styrofoam for 1 day.

    Transfer to liquid nitrogen.

    Freezing

    Freezing kdorfman Mon, 07/09/2012 - 19:59

    Approximately 1 freezing vial per 25 cm2 flask.

    3 vials from large size culture flask.

    Feeding Cells before freezing

    • When cells are 90% confluent (~4 days), replace medium
    • Draw out old medium, replace with 5 mL new (warm) medium.

    Freezing cells

    • Make freezing medium (15% DMSO, 20% serum) in advance
    • Remove medium from flask
    • Rinse once with ~3 mL sterile PBS. (Squirt in, then suck out.)
    • Add 10 – 15 drops trypsin (0.5 – 0.75 mL)
    • Incubate (37C) 5 – 30 min
    • Add 3 mL cold medium (regular)
    • Pour into 15 mL centrifuge tube
    • Optional – rinse with another 2 mL, to capture as many cells as possible
    • Spin 5 – 10 min (until supernate is clear and pellet is hard)
    • Pour off supernate (if pellet is solid – otherwise pipette it off)
    • Resuspend in 1 mL freezing medium (pipette up and down)
    • Transfer to freezing vial (should be slightly more than 1 mL)
    • Put on ice
    • Store overnight in -80C
    • Put into liquid nitrogen.

    Fill out the freezing log

    Frozen cell lines

    Nucleofection

    Nucleofection kdorfman Tue, 09/25/2012 - 18:08

    Mirus

    Mirus kdorfman Fri, 12/06/2019 - 14:36

    general summary

    100 uL Mirus reagent
    2 ug DNA(up to 20 uL DNA solution)
    1 - 5 million cells/mL

    Protocol

    • Warm medium in flask and coverslip-bottom dish
    • Mix DNA and Mirus reagent; warm up
    • Trypsinize cells:
      • remove medium
      • rinse with warm PBS
      • add 0.5 mL trypsin
      • incubate till all cells are in suspension (at least 3 min)
      • add 1 mL COLD medium
      • mix well by pipetting up and down
    • transfer all to sterile 2 mL tube
    • spin 500 x g 3 minutes
    • resuspend cells in Mirus/DNA solution
    • put all into sterile cuvette, close
    • Put cuvette into Nucleofector
    • Choose program1
    • Press the button
    • Use special dropper to transfer one drop to the coverslip dish and the rest to the waiting flask.

    Mirus nucleofection kit


    1. Cell type Program #
      B16 P-031
      3t3 U-030
      HeLa I-013
      LLCPk-1 X-001

      ↩︎

    Cell Density

    Cell Density kdorfman Wed, 09/29/2021 - 16:47
    If cells are % confluent
    in a sq cm flask
    there are million cells in the flask

    selection of nucleofected cells

    selection of nucleofected cells kdorfman Thu, 10/03/2019 - 20:47

    Addgene plasmid selection

    Addgene plasmid selection kdorfman Sat, 05/30/2020 - 13:52

    When cells are medium-light, replace medium with 0.5 g/L G418 (= 0.5 mg/mL)

    Next day, replace with medium with 1 g/L (=1 mg/mL)

    Next day, 2 g/L (=2 mg/mL)

    Solution is 50 mg/mL

    Dilute 10 uL per mL to get 0.5 mg/mL

    Dilute 20 uL per mL to get 1 mg/mL

    Dilute 40 uL/mL to get 2 mg/mL

    Making a Stable Cell Line Expressing Your Favorite Gene

    Making a Stable Cell Line Expressing Your Favorite Gene margaret Fri, 10/21/2011 - 14:38
    1. Make a death curve. This is to determine the minimal concentration of antibiotic that kills cells that do not have the resistance gene.
    2. transfect the cells with the plasmid containing the gene that you want to express in the cells (see Amaxa protocol).
    3. incubate the cells for about 48 hours after transfection so that the gene is expressed, and the resistence gene is expressed.
    4. add the appropriate dose of antibiotic (this is the selection step). The cells that do not contain the resistance gene (i.e. that were not successfully transfected) will die. Change the medium every 2 or 3 days to remove dead cells. Replace medium with fresh medium containing the antibiotic.
    5. split the cells as needed. Use medium with antibiotic. Make some coverslips to see if the cells are expressing the gene of interest.

    After ~2 weeks any cell without the transgene and selectable marker should have died. The remaining cells are a mixed population – different cells might express different levels of the transgene. These can be used for preliminary experiments.

    Making a clonal cell line

  • A Clonal cell line contains cells that are derived from a single cell, and thus are a clone. They will all be genetically identical; this can be very helpful for experiments.
  • a. Trypsinize your 'mixed population' of cells; keep some growing in a flask and freeze some in case your cloning does not work the first time and you need to repeat the procedure.
  • b. take 50ul of the trypsinized cells, dilute again into 1 ml medium. Add 10 -150 ul of this dilute solution of cells to 100mm dishes containing growth medium. Look under the tissue culture scope. The cells should be very dilute – one or just a few cells per field of view. If they are too dense or dilute, adjust accordingly. You want each individual cell to land on the plastic dish and grow into a colony of cells that is separate from other colonies on the dish.
  • c. wait about 1 week for the colonies to grow.
  • d.isolate individual colonies. These are potential clonal cell lines.
    • 1. prepare cloning rings by smearing a small amount of grease on one end; make about 10. Have sterile forceps ready; have trypsin ready. Have a 24 well plate ready with some medium in the number of wells that you are planning to use.
    • 2. remove medium from dish.
    • 3. rinse with PBS--; leave a little bit so the cells don't die.
    • 4. tilt the dish to see the colonies; place a cloning ring over nice large and well separated colonies. Press down firmly. Don't let the ring slide around. Add ~100ul of trypsin to each ring; wait a few mins. Pipette cells up and down (in the ring) add the released cells to one well of a 24 well plate. Each well now has a colony of cells – a potential cell line. When they grow, split each well – place some cells in a well of a six well plate to grow some more and put some in a Matek dish – be sure to label carefully so you know which is which. When the cells in the Matek dish are grown – check them in the fluorescence microscope. If they all have the same level of fluorescence and are healthy looking, they are a potential line (which you can further characterize with a Western Blot to quantify the level of expression).

    Toss any cell lines that are not healthy or have too much or too little fluorescence.

    Lonza Amaxa

    Lonza Amaxa kdorfman Fri, 12/06/2019 - 14:34

    Nucleofection with Lonza Amaxa

    Order VCA-1005 Cell Line Nucleofector Kit L (25 reactions $347)

    Transfection Protocol

    • Warm medium in MatTek dishes to equilibrate CO2
    • Warm additional 0.5 mL medium in sterile microfuge tube
    • Make mucleofection solution, RT, per flask:
      • 19 µL supplement 1
      • 81 µL reagent L for LLCPk (Kit R for 3t3, HeLa)
    • Remove medium from flask
    • Rinse with PBS
    • Add trypsin
    • Incubate ~5 min, or until cells are floating
    • Add cold medium
    • Transfer to sterile 15 mL centrifuge tube
    • Spin 10 min, slow speed
    • Remove medium. Pellet should be soft
    • Loosen pellet by flicking tube
    • Add all 100 µL nucleofection solution plus the DNA (one of the following):
      • 2 µg plasmid DNA
      • 5 µg BAC
      • 5 µL siRNA (20 µM)
    • Pipet up and down to mix
    • Transfer to nucleofection cuvette
    • Insert into nucleofector device, choose program
      • X001 for LLCPK
      • I-013 for HeLa
      • U-030 for 3t3
    • Resuspend cells in 500 µL warm medium
    • Distribute in drops on coverslips of MatTek dishes

    Plating

    Plating kdorfman Mon, 07/09/2012 - 19:50
    • Put medium into dishes
      • MatTek dishes for observations of live cells
      • coverslip in a 35 mm dish for fixation and mounting
    • Let dishes equilibrate in incubator
    • Treat cells in flask with trypsin as for splitting
    • Stop trypsinization with cold medium
    • Add 1 - 4 drops of medium + cells to each dish, depending on cell density
    • Check density of first dish, adjust volume for the remaining dishes
    • Check after 24 hours.

    Splitting

    Splitting kdorfman Mon, 07/09/2012 - 19:44
    • Put 5 mL medium in each flask. 2.5 ml for mini flasks. Label with cell type and passage number
    • Warm flasks in incubator
    • Remove medium from cells.
    • Right away, Rinse once with ~3 mL sterile PBS. (Squirt gently in, Rock the flask, and then suck out. Do not hit the place where cells are. )
    • Add 15 drops sterile trypsin (=~0.75 mL) - enough to cover cells, if more than a thin layer, pull some out
    • Incubate (37C) ~5 min or until all floating, likely no more than 5m.
    • Check to make sure you can see them floating in the liquid
    • Add ~1mL medium to flask, draw up and down to mix.1
      • ~4-8 drops (= 0.2 – 0.4 mL) into each new flask containing medium 23
      • ~4-5 drops ( = 0.2 – 0.25 mL) if cells are 70 – 80% confluent
    • Put back in incubator.

    counting


    1. Use a ratio of medium: trypsin of 2:1 if you need the cells to hang out longer (say for counting) before going into the new flask. ↩︎

    2. if original flask was totally confluent, then use ~1/10th to ~1/5th of the volume; use more if you need cells in a day or two, use less if you don't need cells for a few days. ↩︎

    3. For dishes: if you want a nice monolayer of cells to form within one or two days, add a drop or two of cells AND examine in the microscope. You can quickly get a feel for the number of rounded cells and how heavy the plating will be. You can measure the exact volume and keep track if that helps. Remember to rock to distribute cells on the coverslip or Mattek surface (glass bottom dish). Do not swirl. ↩︎

    Thawing

    Thawing kdorfman Mon, 07/09/2012 - 19:40
    • Label flask with cell line, passage number, date
    • Put 10 mL medium in flask, set in incubator
    • Retrieve frozen vial from LN2 dewar, thaw in incubator or water bath.
    • Put ~1 mL thawed cells into a single flask
    • 24 hours later, replace medium (5 mL)
    • Split as needed

    Labs 2012 & before

    Labs 2012 & before kdorfman Thu, 09/26/2013 - 14:51

    1.1: intro to microscopes

    1.1: intro to microscopes kdorfman Tue, 12/27/2011 - 21:09

    1.2: image formation

    1.2: image formation kdorfman Tue, 12/27/2011 - 21:10

    2.1: Numerical aperture

    2.1: Numerical aperture kdorfman Tue, 12/27/2011 - 21:10

    need dapi slides - can use the same ones for 2.2

    squuze bottle of ethanol to clean oil off lenses.

    Stage micrometer:

    10X: 2.162 pixels/µm

    40X: 8.728 pixels/µm

    Reticle is 2 mm

    Major divisions are 100 µm

    tiniest divisions are 10 µm

    2.2: Resolution

    2.2: Resolution kdorfman Tue, 12/27/2011 - 21:11

    3.1: Fluorescence

    3.1: Fluorescence kdorfman Tue, 12/06/2011 - 15:33

    Fluorescence Microscopy

    LLCPK fixed and stained with DAPI.

    Prepare before the semester starts, keep in a slide box in the refrigerator.

    Bring to room temperature in the morning of the lab to minimize condensation on coverslip.

    Keep track of which number slides have been used, so they will not be used later in any lab where photobleaching is an issue.

    3.2: Immunofluor

    3.2: Immunofluor kdorfman Wed, 11/23/2011 - 16:55

    Immunofluorescence Labeling

    Turn on the Incubator

    Reagents

    • PBS (warm) for rinsing medium off cells before fixation

    • PBS-Tw-Az for rinsing coverslips (in carboy)

    • 2% BSA in PBS to make antibodies

    • Fixative

      • paraglut for students
      • methanol in freezer for certain antibodies
    • Slides

    • Nail polish

    Mystery Antibodies for 2012

    No. Antibody from fixative secondary filter cube
    1 Golgi KD formaldehyde goat anti mouse G
    2 LAP2 PW methanol goat anti mouse G
    3 gamma tub PW paraglut or form goat anti rabbit G
    4 ZO1 KD methanol or paraform goat anti rabbit G
    5 vinculin PW formaldehyde goat anti mouse G

    Cells for 3.2

    Cells for 3.2 kdorfman Tue, 12/06/2011 - 15:01

    Immunofluorescence Labeling

    3.3: Direct fluor

    3.3: Direct fluor kdorfman Tue, 12/06/2011 - 15:57

    Direct Fluorescent Labeling of Organelles


    CHECK THIS FOR HBS vs PBS!!


    Cells

    3t3, LLCPk

    • Fixed in paraglut for phalloidin
      • 8 3t3 (can be fixed as late as the morning of lab)
      • 8 LLCPk (can be from pre-semester prep)
    • Live on small diameter MatTek dishes for Mitotracker and ceramide
      • 8 3t3
      • 8 LLCPk

    Reagents

    3.4 GFP-tags

    3.4 GFP-tags kdorfman Tue, 09/25/2012 - 17:47

    Live cells on MatTek dishes:

    Task 2. GFP-alpha-tubulin cells (everybody) 8 dishes

    Task 3. Nucleofected with GFP version of (2 different ones per group) 4 - 8 dishes each:

    • Rab11
    • E cadherin
    • H2B (GFP)
    • alpha actinin

    Lipofectamine

    Lipofectamine kdorfman Tue, 09/25/2012 - 17:56

    Lipofectamine2000 Plasmid Transfection

    24-48 hrs before class,

    • transfect cells (at 40-60% confluency)
    • Medium without
      • antibiotic
      • serum
      • L-glutamine

    Protocol

    • Dilute DNA with DMEM
    • Dilute lipofectamine2000 with DMEM, incubate <5 min at RT
    • Mix DNA with Lipofectamine2000, incubate >15 min at RT
    • Rinse 2x with DMEM
    • Add complexes to cells, incubate 4 hr 37C
    • Rinse 2x with warm PBS
    • Add normal medium back to cells

    • Per 35 mm dish (1 coverslip):

      • 5 µg DNA in 500 µL
      • 10 µL lipofectamine in 500 µL

    Plasmids

    Plasmids kdorfman Tue, 09/25/2012 - 21:03

    Addgene plasmids have G418 (neomycin) resistance except 26737

    gene fluorophore resistance mg/mL (Abs 280) mg/mL (260/280) µL to get 2 µg source
    rab11 WT GFP kan 6.3 7.8 0.6 Addgene 12674
    E cadherin EGFP amp 2.5 2.5 1 PW
    H2B m cherry kan 1.8 1.3 PW
    H2B EGFP kan 5 7.3 0.33 PW
    EB1 EGFP kan 0.662 0.86 too low PW
    Actin EGFP kan 5.2 4.97 0.4 PW
    a-actinin EGFP kan 1.29 2.73 1 PW
    life act GFP kan (G418 for transfected selection - 1mg/mL for 3t3) 99.1 ng/uL 280: 1.054 260: 1.983 Addgene 58470
    H2B mCherry Amp Addgene 20972
    UtrCH GFP Amp Addgene 26737
    E-cadherin GFP Amp Addgene 28009
    Sec61 beta mcherry Kan Addgene 49155
    Lifeact 7 mcherry Kan Addgene 54491
    Golgi 7 mcherry Kan Addgene 55052
    LaminA-C-18 mcherry Kan Addgene 55068
    Lysosomes-20 mcherry Kan Addgene 55073
    Mito-7 DsRed2 Kan Addgene 55838
    Vimentin-7 EGFP Kan Addgene 56439
    Sec61b-C1 mEmerald Kan Addgene 90992

    Making plasmids

    • PrepEase Endotoxin-Free Maxi Plasmid Kit, 78730 from USB

    • Streak cells on LB-30 µg/mL Kan or LB-amp 50 µg/mL (?)

    • Pick a single colony.

    • Inoculate 100 mL LB (+ 50 – 100 µg/mL ampicilin or 10 – 50 µg/mL kanamycin)

    • Shake overnight, 37C

    • Divide entire culture into 3 or 4 50-mL conicals

    • Use the appropriate rotor in the biochemistry floor model centrifuge.

    • Follow PrepEase directions, except: let filtrate drip directly into column (Steps 5-6)

    • Test in spectrophotometer at 1:500. Adjust dilution as needed

      • Abs 260
      • 260/280 ratio

    DNA concentration

    DNA concentration kdorfman Fri, 10/01/2021 - 19:22
    If your plasmid DNA is ng/uL (=ug/mL)
    and you need ug DNA
    useuL DNA solution

    4.1: Cell cycle

    4.1: Cell cycle kdorfman Mon, 11/14/2011 - 21:58

    Cell Cycle

    Fixed and mounted coverslips of LLCPk cells, stained with DAPI and anti-tubulin

    • normal

    • serum starved

    Cells won't adhere to the coverslip without serum, so grow them first for 24 hours in regular medium.

    Remove the medium, replace with serum-free medium

    Fix the coverslips 24 hours later.

    • Nocodazole treated

    5.1: Motility

    5.1: Motility kdorfman Mon, 11/14/2011 - 21:57

    Motility

    Live cells on MatTek dishes

    8 MatTek dishes each:

    • 3t3

    • B16 (do more than one set, at different concentrations)

    • LLCPK actin

    Must be plated lightly for motility.

    Scheduling

    • Thaw LLCPK-actin, B16 24 hours before plating

    • Plate on all cells (lightly) Monday for Wednesday lab

    3t3’s must have 36-48 hours to stretch out.

    If they get overgrown (especially B16s), scrape some off

    Reagents

    Aliquot non-CO2, serum-free medium

    5.2: Motility Projects

    5.2: Motility Projects kdorfman Wed, 10/12/2011 - 02:36

    CHECK CYTOCHALASIN STOCKS FOR 2012

    2012: 500 nM (250 nM final in dish) should give an effect without causing massive detachment of cells

    Stock is 10 mM in DMSO.

    Need 6 1 mL aliquots of 500 nM in non-CO2 medium:

    • 1 µL 10 mM stock
    • 20 mL medium

    2011: working concentration of cytochalasin D: 1-10uM

    Make 200uM stock for students to dilute with non-CO2 medium

    4 groups using cytochalasin, each doing 4 dishes.

    Maximum conc 10 µM, in 2 mL

    vi = (10 µM * 2 mL)/200 µM = 0.1 mL

    So each group would use at most 0.4mL


    Stock was 10 µL of 10 mM cytochalasin D in DMSO.

    Adding 450 µL of non-CO2 medium makes it 500 µL of 200 µM
    cf = (10 mM * 10 µL)/500 µL = 0.2 mM = 200 µM


    Frozen stock is 2.5 mM
    Invitrogen PHZ1063

    Make 500 µL of 0.2mM
    vi = (0.2mM * 500 µL)/2.5 mM = 40 µL
    Add 460 µL non-CO2 medium

    40 µL aliquots. Each can make 500 µL of 200 µM for use.


    Nocodazole


    Taxol


    6.1: Endocytosis

    6.1: Endocytosis kdorfman Tue, 10/11/2011 - 22:33

    Receptor-mediated Endocytosis

    Monday 10/17/11

    Materials for 6.1

    Materials for 6.1 kdorfman Thu, 10/13/2011 - 18:54
    • fine forceps to pick up coverslips
    • humid chamber petri dishes + filter paper (4 per group)
    • ice in ice bucket
    • small beakers to rinse coverslips
    • glass slides
    • mounting medium
    • nail polish

    cells & reagents 2011

    cells & reagents 2011 kdorfman Wed, 10/10/2012 - 21:07

    Cells on coverslips

    Cells on coverslips kdorfman Tue, 10/11/2011 - 22:37

    For lab on Monday 10/16/11

    Each group gets 4 coverslips of a given type of cell, either 3t3 or LLCPk.

    On Friday 10/14/11: Prepare 32 tiny petri dishes, each with 1 sterile coverslip.

    On Saturday 10/15/11: Plate:

    • 16 coverslips 3t3
    • 16 coverslips LLCPk

    Reagents for 6.1

    Reagents for 6.1 kdorfman Tue, 10/11/2011 - 22:38

    If you used 10 mM Fe-citrate, instead of 89.4 mM, the calculations would be easier.

    The 89.4 value is because Tobias Baskin's lab makes a 89.4 µM Fe-citrate medium, and it's easy to mix up a batch from the 1000x stock solution.

    For convenience, all staining, incubation, etc. will be done in HBS.

    Each group does 4 coverslips, all of the same cell type. Some will use LLCPk, others will use 3t3.

    • HBS to mix everything else in. There is a bottle of sterile 10X on the prep room bench.

      • 6 mL aliquots COLD (final rinse before fix coverslips 1-3)
      • 15 mL aliquots RT in hood (to rinse off fixative)
      • Need ~300 mL total
    • Fe-HBS for coverslips 1-3

      • 20 mL aliquots COLD to rinse off growth medium, then tf
      • 5 mL aliquots WARM (incubation after tf treatment)
      • need some to make Fe-HBS-BSA for transferrin
      • make 200 mL
    Solution stock conc final conc 25 50 75 100 200 mL
    10X HBS 10 1 2.5 5 7.5 10 20 mL
    Fe-citrate (mM) 89.4 0.1 27.96 55.92 84 112 224 µL

    plus water to final volume

    • Fe-HBS-BSA 1 mg/mL to make transferrin solution

      • 5 mg BSA in 5 mL Fe-HBS
    • high-Fe-HBS for coverslip 4

      • 2 mL aliquots COLD
      • 2 mL aliquots WARM
      • need some to make high-Fe-HBS-BSA for transferrin
      • Make 50 mL
    Solution stock conc final conc 10 15 20 25 30 50 mL
    10X HBS 10 1 1 1.5 2 2.5 3 5 mL
    Fe-citrate (mM) 89.4 2 223.7 335.6 448 559.2 671 1118.4 µL

    plus water to final volume

    • High-Fe-HBS-BSA 1 mg/mL to make transferrin solution

      • 5 mg BSA in 5 mL High-Fe-HBS
    • Transferrin in Fe-HBS-BSA to treat coverslips 1-3.

      • Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
      • Each group does 3 50-µL treatments (need extra for pipetting error)
      • Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
      • Aliquot 165 µL
        if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
    # aliquots 3 6 9 12 15 18 21 24
    vi (Tf) 2.64 5.28 7.92 10.56 13.2 15.84 18.48 21.12 µL
    HBS 162.4 324.7 487.1 649.4 811.8 974.2 1136.5 1298.9 µL
    vf 165 330 495 660 825 990 1155 1320 µL
    • Transferrin in high-Fe-HBS-BSA
      • Each group does 1 50 µL treatment.
      • Make 0.44 mL: 7 µL AF-488-Tf + 0.433 mL high-Fe-HBS-BSA
      • Aliquot 55 µL
    # aliquots 1 2 3 4 5 6 7 8
    vi (Tf) 0.88 1.76 2.64 3.52 4.4 5.28 6.16 7.04 µL
    HBS 54.1 108.2 162.4 216.5 270.6 324.7 378.8 433.0 µL
    vf 55 110 165 220 275 330 385 440 µL
    • non-CO2 medium ICE COLD USE Fe-HBS - change manual !!!

      • 3 rinses per coverslip
      • 15 mL aliquots in the refrigerator
      • need over 100 mL
    • HBS-paraformaldehyde 3.7%

      • Need 60 mL
      • Divided in 2 aliquots, one per fume hood
        Ingredient units 10 15 20 25 40 50 60
        paraformaldehyde g 0.37 .555 2 0.925 1.48 1.85 2.22
        water mL 9 13.5 18 22.5 36 45 57.78
        10X HBS mL 1 1.5 2 2.5 4 5 6

    heat paraformaldehyde in water, then add 10X HBS
    Add a drop of 10N NaOH
    Heat and stir in the fume hood

    cells & reagents 2012

    cells & reagents 2012 kdorfman Wed, 10/10/2012 - 21:07

    Cells for 6.1

    Cells for 6.1 kdorfman Wed, 10/10/2012 - 21:15

    Cells for 6.1

    3t3 on coverslips

    3 dishes per group, plus extras = 24

    Plate 2 days before lab.

    Reagents 2012

    Reagents 2012 kdorfman Wed, 10/10/2012 - 21:16

    Omit the high-iron treatment

    For convenience, all staining, incubation, etc. will be done in HBS.

    Each group does 3 coverslips of 3t3.

    • HBS to mix everything else in. There is a bottle of sterile 10X on the prep room bench.

      • 6 mL aliquots COLD (final rinse before fix coverslips 1-3)
      • 15 mL aliquots RT in hood (to rinse off fixative)
      • Need ~300 mL total
    • Fe-HBS

      • 20 mL aliquots COLD to rinse off growth medium, then tf
      • 5 mL aliquots WARM (incubation after tf treatment)
      • need some to make Fe-HBS-BSA for transferrin
      • make 200 mL
    Solution stock conc final conc 25 50 75 100 200 mL
    10X HBS 10 1 2.5 5 7.5 10 20 mL
    Fe-citrate (mM) 89.4 0.1 27.96 55.92 84 112 224 µL

    plus water to final volume

    • Fe-HBS-BSA 1 mg/mL to make transferrin solution
      • 5 mg BSA in 5 mL Fe-HBS
    Solution stock conc final conc 10 15 20 25 30 50 mL
    10X HBS 10 1 1 1.5 2 2.5 3 5 mL
    Fe-citrate (mM) 89.4 2 223.7 335.6 448 559.2 671 1118.4 µL

    plus water to final volume

    • Transferrin in Fe-HBS-BSA
      • Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
      • Each group does 3 50-µL treatments (need extra for pipetting error)
      • Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
      • Aliquot 165 µL
        if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
    # aliquots 3 6 9 12 15 18 21 24
    vi (Tf) 2.64 5.28 7.92 10.56 13.2 15.84 18.48 21.12 µL
    HBS 162.4 324.7 487.1 649.4 811.8 974.2 1136.5 1298.9 µL
    vf 165 330 495 660 825 990 1155 1320 µL
    • **ICE COLD Fe-HBS **

      • 3 rinses per coverslip
      • 15 mL aliquots in the refrigerator
      • need over 100 mL
    • HBS-paraformaldehyde 3.7%

      • Need 60 mL
      • Divided in 2 aliquots, one per fume hood
    Ingredient units 10 15 20 25 40 50 60
    paraformaldehyde g 0.37 .555 2 0.925 1.48 1.85 2.22
    water mL 9 13.5 18 22.5 36 45 57.78
    10X HBS mL 1 1.5 2 2.5 4 5 6

    heat paraformaldehyde in water, then add 10X HBS
    Add a drop of 10N NaOH
    Heat and stir in the fume hood

    6.2: Bulk endocytosis

    6.2: Bulk endocytosis kdorfman Mon, 10/17/2011 - 17:05

    Bulk-phase Endocytosis

    Cells for 6.2 2012

    Cells for 6.2 2012 kdorfman Fri, 10/19/2012 - 12:32

    3t3

    2 coverslips per group

    2 small diameter MatTeks per group

    Cells for 6.2

    Cells for 6.2 kdorfman Mon, 10/17/2011 - 17:09

    Lab is Monday 10/24/11

    cells on coverslips: Students should use the same cell type as for Lab 6.1

    • 3t3 8 (2 per group for 4 groups)
    • LLCPk "

    cells on MatTek dishes
    (small diameter, so 100 µL will be enough to treat the cells):

    • 3t3 8 (2 per group for 4 groups)
    • LLCPk "

    Reagents for 6.2

    Reagents for 6.2 kdorfman Mon, 10/17/2011 - 20:29

    Monday 10/24

    Students use the same cell type as for Lab 6.1

    PBS for rinsing live cells WARM
    PBS for rinsing fixed cells RT
    Take from the supply of sterile aliquots

    Growth media for incubation WARM
    10 mL aliquots each

    • F10-Hams for LLCPk cells
    • DMEM for 3t3 cells
    • non-CO2 medium for observing live cells (could use this for all cells for simplicity's sake)

    TMR-dextran to stain endosomes in cells on coverslips
    Invitrogen D3308, 10 mg
    Make 10 mg/mL stock (add 1 mL to the bottle)

    Make 8.8 µL aliquots in 2 mL tubes.

    TMR-dextran Working concentration = 0.05 mg/mL in medium
    2 coverslips at 50 µL/coverslip (=~110 µL/group)

    Add 1751.2 µL medium to 8.8 µL 10mg/mL stock to make 0.05 mg/mL working solution.
    Aliquot ~105 µL/group

    #aliquots 1 2 3 4 5 6 7 8
    vi (10 mg/mL TMR-dex) 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 µL
    medium 218.9 437.8 656.7 875.6 1094.5 1313.4 1532.3 1751.2 µL
    vf 220 440 660 880 1100 1320 1540 1760 µL

    TMR-dextran/ FITC-dextran to stain endosomes in cells in MatTeks
    tmr as above

    FITC-dextran Working concentration = 0.5 mg/mL in medium
    (1mg/mL might be better, but that uses up a whole bottle.)
    2 dishes at 100 µL/dish (=~220 µL/group)

    Add 1742.4 µL medium to an 8.8 µL aliquot of 10 mg/mL TMR-dex plus 8.8 µL 100 mg/mL FITC-Dex

    #aliquots 1 2 3 4 5 6 7 8
    vi (10 mg/mL TMR-dex) 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 µL
    vi (100 mg/mL FITC-dex) 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 µL
    medium 217.8 435.6 650.1 871.2 1089 1306.8 1524.6 1742.4 µL
    vf 220 440 660 880 1100 1320 1540 1760 µL

    Methylamine solution (1M)
    Sigma 426466-100mL 40% w/vol (=12.9M; MW=31) ~$25
    (Working concentration ~20mM - ~20µL/mL. Students need ~100 µL)

    Make 1 mL, aliquot ~120 µL

    Solution stock conc final conc 1 5 10 15 20 mL
    10X PBS 10 1 0.1 0.5 1 1.5 2 mL
    methylamine 12.9 1 78 388 775 1163 1550 µL

    plus water to final volume


    Fixative

    • PBS-paraformaldehyde 3.7%
      • Need 70 mL
      • Divided in 2 aliquots, one per fume hood
    Ingredient 10 15 20 25 40 50 60 70 mL final
    paraformaldehyde 0.37 0.555 2 0.925 1.48 1.85 2.22 2.59 g
    water 9 13.5 18 22.5 36 45 57.78 63 mL
    10X PBS 1 1.5 2 2.5 4 5 6 7 mL

    heat paraformaldehyde in water, then add 10X HBS
    Add a drop of 10N NaOH
    Heat and stir in the fume hood

    OR

    Ingredient 10 15 20 25 40 50 60 70 mL final
    32% paraformaldehyde 1.16 1.73 2.31 2.89 4.63 5.78 6.94 8.09 mL
    10X PBS 1 1.5 2 2.5 4 5 6 7 mL
    water 7.84 11.77 15.69 19.61 31.38 39.22 47.06 54.91 mL

    6.3: Endosome txport

    6.3: Endosome txport kdorfman Wed, 10/19/2011 - 18:44

    Transport of Endosomes along Microtubules

    Wednesday 10/26/11

    Cells for 6.3

    Cells for 6.3 kdorfman Wed, 10/19/2011 - 18:47

    LLCPk-GFP-tub

    3 small diameter MatTek dishes per group

    Plate 24 dishes 2 days before lab

    Reagents for 6.3

    Reagents for 6.3 kdorfman Wed, 10/19/2011 - 18:48

    HBS-BSA 1mg/mL

    • to mix transferrin ( 3 mL) and nocodazole (15 mL) in

    • to rinse growth medium off before and after treatment. 6 mL per dish x 3 dishes per group x 7 groups = 126mL. Aliquot 20 mL.

    • make 200 mL

    ingredient 10 20 25 30 50 75 100 200 250 mL
    10 x HBS 1 2 2.5 3 5 7.5 10 20 25 mL
    BSA 0.01 0.02 0.025 0.03 0.05 0.075 0.1 0.2 0.25 g

    plus water to final volume


    Fe-HBS-BSA
    to mix transferrin in
    Need 2.3 mL; make 3 mL

    Solution stock conc final conc 1 2 2.5 3 4 mL
    Fe-citrate (mM) 89.4 0.1 1.118 2.237 2.796 3.35 4.47 µL

    in Fe-HBS-BSA to final volume


    Transferrin in Fe-HBS-BSA
    Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD)
    stock is 5 mg/ml (=62.5 µM).
    Final concentration = 1 µM.

    • Each group may do up to 3 100 µL treatments (= 3 x 7 x 105 µL = 2.205 mL)
    • Make 105 µL aliquots (21)
    • Make 2.3 mL: 36.8 µL AF-488-Tf + 2.263 mL Fe-HBS-BSA

    if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.

    # aliquots 1 2 3 6 9 12 15 18 21
    vi 62.5µM tf 1.68 3.36 5.04 10.1 15.1 20.2 25.2 30.2 35.3 µL
    HBS-Fe-BSA 103.3 206.6 310 620 930 1240 1550 1860 2170 µL
    Vf 1 µM tf 105 210 315 630 945 1260 1575 1890 2205 µL

    Nocodazole
    Acros 358240100, 10 mg
    1µM in HBS-BSA
    (Working concentration range is usually 100nM - 30 µM.)
    Stock is 10 mg/mL (=33mM)
    (Add 1mL DMSO to 10 mg in bottle = 33mM. Aliquot 3.03 µL. To make 1mM, add 97 µL.)

    Make enough for each group to do two 1 mL treatments.
    Aliquot 1.01 mL per tube, 15 tubes = 15 mL. Make 16 mL

    • Add 97 µL HBS-BSA to 3 µL 33mM in tube to make 1 mM
    • Mix 1 part 1mM with 999 parts HBS-BSA to make 1µM: 16 mL + 1.6 µL
    # aliquots 1 2 3 4 5 10 15
    vi 1 mM noc 1.01 2.02 3.03 4.04 5.05 10.1 15.15 µL
    HBS-BSA 1.009 2.018 3.027 4.036 5.045 10.09 15.135 mL
    vf 1 µM noc 1.01 2.02 3.03 4.04 5.05 10.1 15.15 mL

    non-CO2 medium
    to mix TMR-dextran in
    to incubate cells in


    TMR-dextran

    in non-CO2 medium
    Each group may do up to 3 treatments = 3 x 7 x 100 µL = 2.205 mL.

    Invitrogen D3308, 10 mg
    Make 100 mg/mL stock (add 100µL to the bottle)
    1 µL aliquots

    TMR-dextran Working concentration = 0.05 mg/mL in medium
    100 µL per treatment
    Aliquot 105 µL, make enough for each group to do 3

    Make 10 mg/mL dilution to make student aliquots
    1 µL 100 mg/mL stock + 9µL medium

    no. aliquots 1 2 3 6 9 12 15 18 21
    vi (10 mg/mL TMR-dex) 0.525 1.05 1.575 2.2 3.15 4.2 6.3 7.875 11.025 µL
    medium 104.5 128.9 656.7 875.6 1094.5 1313.4 1532.3 1751.2 µL
    vf (0.05 mg/mL TMR-dex) 105 210 315 630 945 1260 1575 1890 2205 µL

    7.2: Calcium

    7.2: Calcium kdorfman Wed, 10/26/2011 - 19:05

    Release of Internal Calcium Stores

    Wednesday, 11/2/11

    Rescheduled for Monday, 11/7/11, due to snowday 10/31/11

    7.1: External stimuli

    7.1: External stimuli kdorfman Wed, 10/26/2011 - 16:08

    Cellular Responses to External Stimuli
    Monday, 10/31/11

    Rescheduled for 11/2/11 because of storm on 10/31/11

    Cells for 7.1

    Cells for 7.1 kdorfman Wed, 10/26/2011 - 16:12

    3t3 on large diameter MatTek dishes

    4 dishes per group plus extra = 32

    Plate 2 days before class.

    Reagents for 7.1

    Reagents for 7.1 kdorfman Wed, 10/26/2011 - 16:13

    HBS

    HBS kdorfman Wed, 10/26/2011 - 16:15

    HBS for washes and making solutions

    25 mL per group

    (total = 175 mL)

    Make from 10X

    Make 350 mL

    Pluronic F-127

    Pluronic F-127 kdorfman Wed, 10/26/2011 - 16:26

    Stock

    comes as dry granules - store on shelf

    Also comes as solution Fisher 50-310-493
    PROMOCELL GMBH PLURONICF-127 20%IN DMSO 1ML $44

    Make 0.5 mL with 0.1g (= 200 mg/mL =20% w/v) in DMSO

    10 µL aliquots - each makes 10 mL 0.02%

    Store in freezer.

    ATP

    ATP kdorfman Wed, 10/26/2011 - 17:13

    10 mM ATP stock (from Maniotis)

    60 mg ATP in 8 mL H2O
    pH to 7.0 with 0.1 M NaOH
    Bring volume to 10 mL with H2O

    Aliquot and freeze.


    10 µM solution for use (=5.731 mg/L) in HBS

    Make enough for Labs 7.1 and 7.2:
    Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
    Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

    Make 40 mL
    40 µL 10 mM ATP stock in 40 mL HBS (1 µL 10 mM ATP stock per 1 mL HBS.)

    Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2

    Bradykinin

    Bradykinin kdorfman Wed, 10/26/2011 - 17:22

    1 mg/mL stock
    (= 1.0602 mM) Sigma B3259 - 1 mg septum bottle
    MW = 1060.2
    Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)

    Aliquots are 21.2 µL.

    2 µM solution for use
    (=2.12 µg/mL)

    NOTE: Rather weak response in 2011. Try doubling the concentration?

    Make enough for Labs 7.1 and 7.2:

    • Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
    • Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

    Make 40 mL

    ~2 µL 1 mg/mL stock per 1 mL working solution

    Each 21.2 µL aliquot of 1 mg/mL makes 10 mL 2µM working solution

    84.8 µL 1 mg/mL stock in 40 mL HBS

    Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2)


    4 µM solution for use
    (=4.24 µg/mL)

    NOTE: Double the 2011 concentration

    Make enough for Labs 7.1 and 7.2:

    • Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
    • Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

    Make 40 mL

    ~4 µL 1 mg/mL stock per 1 mL working solution

    Each 21.2 µL aliquot of 1 mg/mL makes 5 mL 2µM working solution

    169.6 µL 1 mg/mL stock in 40 mL HBS (8 aliquots)

    Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2)

    Fluo-4

    Fluo-4 kdorfman Wed, 10/26/2011 - 16:22

    Fluo-4 stock
    Invitrogen F14217 500 µL
    1mM in DMSO
    Protect from light
    Store in dissicator.
    20 µL aliquots. Each makes 10 mL of 2 µM solution

    Fluo-4 staining solution
    2 µM Fluo-4 + 0.02% pluronic in HBS

    Make enough for 7.1 and 7.2: 250 µL x 4 expts x 8 grps x 2 days = 16 mL.
    Make 20 mL
    Need 16 aliquots, each ~1.05 mL

    ingredient 5 10 15 20 mL
    1 mM Fluo-4 stock 10 20 30 40 µL
    20 % w/v Pluoronic 5 10 15 20 µL

    plus HBS to final volume

    Vasopressin

    Vasopressin kdorfman Wed, 10/26/2011 - 18:29

    1 mM stock in water
    (NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)

    1 mg in bottle, MW = 1084
    Add 0.92 mL to make 1mM stock

    1 µL aliquots in freezer


    1 µM dilution to make student solutions

    1 µL 1 mM in 999 µL water

    (NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)


    10 nM (= 1.084 µg/mL) for students

    NOTE: Rather weak response in 2011. Try doubling the concentration?

    1 part 1µM dilution to 99 parts HBS

    Make enough for Labs 7.1 and 7.2: Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

    Make 40 mL: 400 µL 1µM Vasopressin + 39.6 mL HBS

    Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2

    test drop downs for 7.1 reagents

    test drop downs for 7.1 reagents kdorfman Thu, 10/27/2011 - 16:55

    This page tests the use of collapsible sections for recipe pages

    Cells for 7.2

    Cells for 7.2 kdorfman Wed, 10/26/2011 - 19:07

    3t3 on large-diameter MatTek dishes

    4 dishes per group, plus extra = 32

    Plate on Monday before Wednesday lab, or Saturday if it's on Monday

    Reagents for 7.2

    Reagents for 7.2 kdorfman Wed, 10/26/2011 - 19:08

    Use left-over aliquots of stimulants from 7.1 with calcium, make calcium-free reagents for 7.2

    ATP

    ATP kdorfman Wed, 10/26/2011 - 19:23

    10 mM ATP Stock
    (from Maniotis)

    60 mg ATP in 8 mL H2O
    pH to 7.0 with 0.1 M NaOH
    Bring volume to 10 mL with H2O

    Aliquot and freeze.


    10 µM (=5.731 mg/L) ATP in HBS

    Use ATP aliquots from Lab 7.1


    10 µM ATP, Calcium-free

    Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

    Make 13 mL
    13 µL 10 mM ATP stock in 13 mL ca- HBS (1 µL 10 mM ATP stock per 1 mL HBS.)

    8 1.6 mL aliquots for Lab 7.2

    Bradykinin

    Bradykinin kdorfman Wed, 10/26/2011 - 19:25

    1mg/mL Bradykinin stock
    (= 1.0602 mM) Sigma B3259 - 1 mg septum bottle
    MW = 106.02
    Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)


    2 µM Bradykinin in HBS

    Use bradykinin aliquots from Lab 7.1


    2 µM Bradykinin, Calcium-free
    (=2.12 µg/mL)

    Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

    Frozen aliquots are 21.2 µL 1 mg/mL. Makes 10 mL

    ~2 µL 1 mg/mL stock per 1 mL working solution

    21.2 µL 1 mg/mL stock in 10 mL Ca-free HBS

    Make 6 aliquots of 1.6 mL

    Make more if necessary. (Aliquots should have been enough for 15 mL?)

    Fluo-4

    Fluo-4 kdorfman Wed, 10/26/2011 - 19:17

    Use Fluo-4 aliquots from Lab 7.1

    HBS

    HBS kdorfman Wed, 10/26/2011 - 19:14

    HBS needed for rinsing and to make other solutions

    (3 rinses + 1 sham dilution) x 4 expts/grp x 1 mL = 16 mL

    Aliquot 25 mL per group

    300 mL HBS

    300 mL Ca-free HBS

    Ionomycin

    Ionomycin kdorfman Wed, 10/26/2011 - 19:41

    Ionomycin stock

    1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.

    3 µM Ionomycin in HBS
    Dilute stock 1:333 in HBS: 3 µL/1 mL

    21 µL in 7 mL, aliquot 800 µL

    They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.

    3 µM Ionomycin in Ca-free HBS
    Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
    39 µL in 13 mL

    Aliquot 1.55 mL so they can do two 750 µL experiments

    Vasopressin

    Vasopressin kdorfman Wed, 10/26/2011 - 19:34

    1 mg/mL (= 0.9225 mM) Vasopressin stock in water


    1 µM dilution to make student solutions

    1 µL 1 mg/mL in 922.5 µL water (NO CALCIUM, so stock can be used for Ca-free solutions )


    10 nM (= 1.084 µg/mL) Vasopressin in HBS for students

    Use Vasopressin aliquots from Lab 7.1


    Calcium Free Vasopressin 10 nM

    10 nM (= 1.084 µg/mL) for students

    1 part 1µM dilution to 99 parts Ca-free HBS

    Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL

    Make 13 mL: 130 µL + 13 mL Ca-free HBS

    Make 8 aliquots of 1.6 mL

    8: Projects

    8: Projects kdorfman Tue, 11/22/2011 - 14:20

    Projects

    Have students make a class poster for the bulletin board in the hall.

    ER-tracker

    ER-tracker kdorfman Tue, 11/22/2011 - 14:22

    ER-Tracker from Invitrogen

    E34250 ER-Tracker™ Red (BODIPY® TR glibenclamide) for live-cell imaging 100 μg

    1mM Stock

    Add 110 µL DMSO to the 100 µg in vial.

    • Aliquot 1 µL

    • Freeze

    1 µM working concentration

    • add 0.999 mL HBSS/Ca/Mg

    Protocol

    • rinse with warm HBSS

    • treat with 1 mL warm 1 µM ER-tracker

    • incubate 15-30 min at 37C

    To view stained cells

    • rinse with medium

    • view with fluorescence microscope

    To fix cells

    • 4% formaldehyde, for 2 min, at 37C

    • rinse 2x in buffer - NO TRITON X-100

    HBSS/Ca/Mg

    HBSS/Ca/Mg kdorfman Wed, 11/23/2011 - 20:08

    Hank's Balanced Salt Solution

    Gibco cat. #14025-092

    To make 1 Liter from dry ingredients

    Components MW (mg/L) mM
    Calcium Chloride (CaCl2) (dihyd.) 147 185 1.26
    Magnesium Chloride (MgCl2-6H2O) 203 100 0.493
    Magnesium Sulfate (MgSO4-7H2O) 246 100 0.407
    Potassium Chloride (KCl) 75 400 5.33
    Potassium Phosphate monobasic (KH2PO4) 136 60 0.441
    Sodium Bicarbonate (NaHCO3) 84 350 4.17
    Sodium Chloride (NaCl) 58 8000 137.93
    Sodium Phosphate dibasic (Na2HPO4-6H20) 268 90.6 0.338
    D-Glucose (Dextrose) 180 1000 5.56

    Filter sterilize



    To make from stock solutions:

    Components Ci (M) cf (mM) 1000 500 250 mL
    CaCl2 0.5 1.26 2.52 1.26 0.63 mL
    MgCl2 1 0.493 0.493 0.247 0.108 mL
    MgSO4 1 0.407 0.407 0.204 0.102 mL
    KCl 1 5.33 5.33 2.67 0.131 mL
    KH2PO4 1 0.441 0.441 0.22 0.11 mL
    NaHCO3 0.5 4.17 8.34 4.17 0.209 mL
    NaCl 5 137.93 27.59 13.79 6.9 mL
    Na2HPO4 1 0.338 0.338 0.169 0.085 mL
    Glucose - - 1 0.5 0.25 g

    Filter sterilize

    Latrunculin B

    Latrunculin B kdorfman Wed, 11/30/2011 - 18:21

    Alexis Biochemicals 350-036-C100 Catalog # t110

    Inhibits actin polymerization and disrupts microfilament organization, as well as microfilament-mediated processes

    Reported to be 10 to 100-fold more potent than the cytochalasins. May act more slowly, though. Lots of variation between cell lines.

    Inactivated by FBS. (Rinse medium off before treatment.)

    MW = 395.5

    Soluble in DMSO and ethanol.

    Store in freezer.

    Active concentration range: 90 nm ( =~0.1µM) to 2.5 µM

    (2.5 µM = 25 x 100 nm)

    Stock is 1mm

    µL 1mm stock µL serum-free buffer or medium µM final concentration
    1 * 99 10
    1 399 2.5
    1 999 1

    *Use this to make further dilutions

    Projects 2012

    Projects 2012 kdorfman Wed, 11/07/2012 - 20:29

    For Wed, 11/14/12

    Names cell type # dish/coverslip reagents
    Jonathan & Laura LL parentals 5 slips fixative, mounting medium
    Mike & Theresa 3t3 4 dishes transferrin, Fe-HBS, HBS
    Mike & Mike LL alpha-GFP, myo-RFP 4 dishes blebbistatin, non-CO2 medium
    Cassie & Marc LL parentals 4 dishes nuc-blue, HBS, Fluo-4, non-CO2
    Dave & Tyler LL alpha-GFP 2 dishes, 2 slips Fix, mitotracker, Rhodamine123, non-CO2, PBS
    Mike & Katherine LL parentals 3 dishes ceramide, non-CO2, PBS
    Ray & Poya LL alpha-GFP 2 dishes non- CO2

    Plating:

    • LL parental: 5 coverslips, 7 dishes
    • LL alphas: 4 dishes, 2 coverslips
    • LL alpha, myo: 4 dishes
    • 3t3: 4 dishes

    Blebbistatin

    Blebbistatin kdorfman Wed, 11/21/2012 - 19:51

    (-)-Blebbistatin purchased from Sigma B05650-1mg

    Info from Cayman:

    A stock solution may be made by dissolving the (±)-blebbistatin in an organic solvent purged with an inert gas. (±)-Blebbistatin is soluble in organic solvents such as DMSO and dimethyl formamide (DMF). The solubility of (±)-blebbistatin in these solvents is approximately 10 mg/mL.

    (±)-Blebbistatin is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, (±)-blebbistatin should first be dissolved in DMF and then diluted with the aqueous buffer of choice. (±)-Blebbistatin has a solubility of approximately 0.5 mg/ml in a 1:1 solution of DMF:PBS (pH 7.2) using this method. We do not recommend storing the aqueous solution for more than one day.

    (±)-Blebbistatin is a selective cell-permeable inhibitor of non-muscle myosin II ATPases.1,2 It rapidly and reversibly inhibits Mg-ATPase activity and in vitro motility of non-muscle myosin IIA and IIB for several species (IC50 = 0.5-5.0 μM), while poorly inhibiting smooth muscle myosin (IC50 = 80 μM).3 Through these effects, blebbistatin blocks apoptosis-related bleb formation, directed cell migration and cytokinesis in vertebrate cells. Blebbistatin is inactivated by UV light,4 which may be particularly important in fluorescent cell imaging applications.

    Note green fluorescent crystals seen in a 75µM solution.

    Stock was 1 mg/mL in DMSO

    44 µL 1mg/mL blebbistatin in DMSO in 1 mL non-CO2 medium

    REDO

    1 mg in 34 µL DMSO = ~100mM

    MW = 292

    Use at ~150 µM

    Cells Monday 11/26

    Cells Monday 11/26 kdorfman Mon, 11/19/2012 - 19:20
    Names cell type dishes coverslips reagents
    Jonathan & Laura LL parentals 5 (Sunday)
    Mike & Theresa 3t3 6
    Mike & Mike LL gamma tub 4
    Cassie & Marc LL parentals 4 fluo4-AM
    Dave & Tyler LL alpha-GFP 2 2
    Mike & Katherine LL parentals 2
    LL alpha 2
    Ray & Poya LL alpha-GFP 8 ciliobrevin (HPI-4) (fix) BI2536

    Plating:

    • LL parental:
      • 5 coverslips,
      • 6 dishes
    • LL gamma: 4 dishes
    • LL alpha:
      • 2 dishes
      • 10 coverslips
    • LL alpha, myo:
    • 3t3: 6 dishes

    Cells Wed 11/14

    Cells Wed 11/14 kdorfman Mon, 11/19/2012 - 19:41

    For Wed, 11/14/12

    Names cell type # dish/coverslip reagents
    Jonathan & Laura LL parentals 5 slips fixative, mounting medium
    Mike & Theresa 3t3 4 dishes transferrin, Fe-HBS, HBS
    Mike & Mike LL alpha-GFP, myo-RFP 4 dishes blebbistatin, non-CO2 medium
    Cassie & Marc LL parentals 4 dishes nuc-blue, HBS, Fluo-4, non-CO2
    Dave & Tyler LL alpha-GFP 2 dishes, 2 slips Fix, mitotracker, Rhodamine123, non-CO2, PBS
    Mike & Katherine LL parentals 3 dishes ceramide, non-CO2, PBS
    Ray & Poya LL alpha-GFP 2 dishes non- CO2

    Plating:

    • LL parental: 5 coverslips, 7 dishes
    • LL alphas: 4 dishes, 2 coverslips
    • LL alpha, myo: 4 dishes
    • 3t3: 4 dishes

    Cells Wed 11/28

    Cells Wed 11/28 kdorfman Mon, 11/26/2012 - 19:48
    Names cell type dishes coverslips reagents
    Jonathan & Laura 0 0 0
    Mike & Theresa 3t3 3 normal and 6 light (for weekend)
    Mike & Mike LL gamma tub 4 Blebbistatin
    Cassie & Marc LL parentals 2 (fluo4-AM)
    Dave & Tyler LL alpha-GFP 2 2
    Mike & Katherine - 0 0
    Ray & Poya 0 0 0

    HPI-4 (ciliobrevin)

    HPI-4 (ciliobrevin) kdorfman Tue, 11/20/2012 - 15:51

    Sigma H4541-5MG

    MW 358.18

    solubility 20 mg/mL

    Make stock in DMSO

    100mM = 5 mg/140 µL

    http://www.ncbi.nlm.nih.gov/pubmed/22425997#

    Use at 100uM. 1 µL/1 mL

    light sensitive

    fix in para glut, to preserve the GFP micro tubules.

    Para formaldehyde may also work

    cells Mon 12/3

    cells Mon 12/3 kdorfman Wed, 11/28/2012 - 20:05
    Names cell type dishes coverslips reagents
    Jonathan & Laura B16 - 5 formaldehyde fix
    Mike & Theresa 3t3 6 0
    Mike & Mike LL gamma tub 4 - (blebbistatin)
    Cassie & Marc LL parentals 4 0
    Dave & Tyler LL actin-GFP 2 -
    Mike & Katherine LL alpha tub 0 3 formaldehyde fix
    Ray & Poya - - -

    Thaw B16 and actins on Thursday 11/29

    Plate all types lightly on Friday 11/30 for Monday

    cells Sat 12/1

    cells Sat 12/1 kdorfman Wed, 11/28/2012 - 20:16
    Names cell type dishes coverslips reagents
    Mike & Theresa 3t3 3 -
    Cassie & Marc LL parentals 6 - (fluo4-AM)
    • Plate on Thursday

    • Also make heavy flasks for Plating on Friday

    cells Wed 12/5

    cells Wed 12/5 kdorfman Mon, 12/03/2012 - 16:36
    Names cell type dishes coverslips reagents
    Jonathan & Laura
    Mike & Theresa 3t3 (light) 4
    Mike & Mike gamma tubulin 4 0
    Cassie & Marc Parental LLCPK1 (heavy) 6 0 HBS!
    Dave & Tyler
    Mike & Katherine
    Ray & Poya

    projects 2011

    projects 2011 kdorfman Wed, 11/07/2012 - 20:15

    Cells for 11/28/11

    Cells for 11/28/11 kdorfman Wed, 11/23/2011 - 18:06
    Group Cells MatTek Coverslip Reagents
    Kevin & Chris LLCPk 0 6 ER tracker, anti golgi
    Megan & Olivia actin 3 (20 mm) 0 cyto D
    Andrew & Amelia actin 4 (20 mm) 0 Cyto D
    Alex & Quentin tubulin 4 (14mm) 0 Noc & taxol
    Luke & Don tubulin 4 (20 mm) 0 ER tracker
    Jon & Silas 3t3 2 (20 mm) 4 Cyto D
    Molly & Jason s2 0 0 Con A, colcemid or colchicine

    Cells for 11/30/11

    Cells for 11/30/11 kdorfman Mon, 11/28/2011 - 15:13
    Group Cells MatTek Coverslip Reagents
    Kevin & Chris LLCPk 4 ER tracker, anti golgi
    Meghan & Olivia actin 7 (20 mm) cyto D, latrunculin
    Andrew & Amelia actin 5 (20 mm) Cyto D
    Alex & Quentin tubulin 4 (14mm) Noc & taxol
    Luke & Don tubulin 4 (20mm) ER tracker, noc
    Jon & Silas 3t3 5 (20mm) Cyto D, ATP
    Molly & Jason s2 Con A, colcemid or colchicine

    Cells for 12/5/11

    Cells for 12/5/11 kdorfman Wed, 11/30/2011 - 21:11
    Group Cells MatTek Coverslip Reagents
    Kevin & Chris LLCPk 4 ER tracker, anti golgi
    Meghan & Olivia actin 5 (20 mm) cyto D, latrunculin
    Andrew & Amelia actin 2 (20 mm) 6 Cyto D
    Alex & Quentin tubulin 4 (14mm) Noc & taxol
    Luke & Don tubulin 4 (20mm) ER tracker, noc
    Jon & Silas 3t3 (20mm) 5 Cyto D, ATP
    Molly & Jason s2 Con A, colcemid or colchicine

    Cells for 12/7

    Cells for 12/7 kdorfman Mon, 12/05/2011 - 17:26
    Group Cells MatTek Coverslip Reagents
    Kevin & Chris LLCPk ER tracker, anti golgi
    Meghan & Olivia actin (20 mm) cyto D, latrunculin
    Andrew & Amelia actin (20 mm) Cyto D
    Alex & Quentin tubulin (14mm) Noc & taxol
    Luke & Don tubulin (20mm) ER tracker, noc
    Jon & Silas 3t3 (20mm) Cyto D, ATP
    Molly & Jason s2 Con A, colcemid or colchicine

    Labs 2013

    Labs 2013 kdorfman Thu, 09/26/2013 - 14:27

    1.1 2013

    1.1 2013 kdorfman Thu, 09/26/2013 - 14:28

    W 9/04

    Intro to Equipment

    1.2 2013

    1.2 2013 kdorfman Thu, 09/26/2013 - 14:28

    W 9/09

    Imaging mitosis with fixed cells. Image Spatial Quantification

    1.3 2013

    1.3 2013 kdorfman Thu, 09/26/2013 - 14:32

    W 9/11

    Image Formation in the Microscope

    2.1 2013

    2.1 2013 kdorfman Thu, 09/26/2013 - 14:33

    M 9/16

    Numerical Aperture

    2.2 2013

    2.2 2013 kdorfman Thu, 09/26/2013 - 14:37

    W 9/18

    Resolution

    3.1 2013

    3.1 2013 kdorfman Thu, 09/26/2013 - 14:38

    M 9/23

    Fluorescence Microscopy

    3.2 2013

    3.2 2013 kdorfman Thu, 09/26/2013 - 14:39

    W 9/25

    Identification of Cellular Organelles

    3.3 2013

    3.3 2013 kdorfman Thu, 09/26/2013 - 14:40

    M 9/30

    Labeling Cellular Structures

    Cells

    • paraglut fixed LL's for students to stain for tubulin (make extra!)

    • paraglut fixed LL's for students to stain for phalloidin

    • paraglut fixed 3t3's for students to stain for phalloidin

    • live LL's to fix in paraglut (2014: treat first with mitotracker, then fix in formaldehyde no detergent)

    Solutions

    Materials

    • forceps

    • 50 mL beakers (switch to holders and 100 mL beakers?)

    • petri dishes for humid chambers

    • filter paper for humid chambers

    • mounting medium

    • slides

    • nail polish

    • kimwipes

    • parafilm

    3.4 2013

    3.4 2013 kdorfman Thu, 09/26/2013 - 14:41

    W 10/2

    Imaging Living Cells Expressing GFP-Tagged Proteins

    2014

    • LL-EB1-GFP stable line

    • LL-alpha tubulin=GFP stable line

    ** transfected**

    • H2B m cherry

    • alpha actininin (GFP?)

    4.1 2013

    4.1 2013 kdorfman Thu, 09/26/2013 - 14:42

    W 10/9

    Motility in Control Cells

    On MatTek dishes:

    • 3t3

    • B16

    • LL-GFP actin

    Non-CO2 medium

    4.2 2013

    4.2 2013 kdorfman Thu, 09/26/2013 - 14:43

    Tu 10/15 & W 10/16

    Mechanism of Motility

    Students check for effect of dose and time on actin filaments. Treat LL parentals with cytochalasin D, then stain actin with phalloidin.

    5.1 2013

    5.1 2013 kdorfman Thu, 09/26/2013 - 14:45

    W 10/23

    Receptor-mediated endocytosis

    NO LL's

    NO high iron

    3 coverslips of 3t3 per group

    Try Lysotracker (Invitrogen L-7528)

    • 50 - 75 nM

    • stock = 1 mM in DMSO

    • 30 min - 2 hours incubation warm.

    • Add lysotracker to cells at 1 pm.

      • Make 100 µM stock
      • 1 µL of 100 µM stock to each dish. Assume ~1.5 mL per dish

    Omit the high-iron treatment

    For convenience, all staining, incubation, etc. will be done in HBS.

    Each group does 3 coverslips of 3t3.

    • HBS to mix everything else in. There is a bottle of sterile 10X on the prep room bench.

      • 6 mL aliquots COLD (final rinse before fix coverslips 1-3)
      • 15 mL aliquots RT in hood (to rinse off fixative)
      • Need ~300 mL total
    • Fe-HBS

      • 20 mL aliquots COLD to rinse off growth medium, then tf
      • 5 mL aliquots WARM (incubation after tf treatment)
      • need some to make Fe-HBS-BSA for transferrin
      • make 200 mL
    Solution stock conc final conc 25 50 75 100 200 mL
    10X HBS 10 1 2.5 5 7.5 10 20 mL
    Fe-citrate (mM) 89.4 0.1 27.96 55.92 84 112 224 µL

    plus water to final volume

    • Fe-HBS-BSA 1 mg/mL to make transferrin solution

      • 5 mg BSA in 5 mL Fe-HBS
    • Transferrin in Fe-HBS-BSA

      • Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
      • Each group does 3 50-µL treatments (need extra for pipetting error)
      • Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
      • Aliquot 165 µL
        if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
    # aliquots 3 6 9 12 15 18 21 24
    vi (Tf) 2.64 5.28 7.92 10.56 13.2 15.84 18.48 21.12 µL
    HBS 162.4 324.7 487.1 649.4 811.8 974.2 1136.5 1298.9 µL
    vf 165 330 495 660 825 990 1155 1320 µL

    Old fashioned way:

    Ingredient units 10 15 20 25 40 50 60
    paraformaldehyde g 0.37 .555 0.74 0.925 1.48 1.85 2.22
    water mL 9 13.5 18 22.5 36 45 57.78
    10X HBS mL 1 1.5 2 2.5 4 5 6

    heat paraformaldehyde in water, then add 10X HBS
    Add a drop of 10N NaOH
    Heat and stir in the fume hood


    OMIT HIGH IRON

    Solution stock conc final conc 10 15 20 25 30 50 mL
    10X HBS 10 1 1 1.5 2 2.5 3 5 mL
    Fe-citrate (mM) 89.4 2 223.7 335.6 448 559.2 671 1118.4 µL

    plus water to final volume


    5.2 2013

    5.2 2013 kdorfman Thu, 09/26/2013 - 14:46

    M 10/28

    Bulk-phase endocytosis

    ** 2 coverslips, 2 small diameter MatTek dishes 3t3

    PBS for rinsing live cells WARM
    PBS for rinsing fixed cells RT
    Take from the supply of sterile aliquots

    Growth media for incubation WARM
    10 mL aliquots each

    • DMEM for 3t3 cells
    • non-CO2 medium for observing live cells (could use this for all cells for simplicity's sake)

    TMR-dextran to stain endosomes in cells on coverslips
    Invitrogen D3308, 10 mg
    Make 10 mg/mL stock (add 1 mL to the bottle)

    Make 8.8 µL aliquots in 2 mL tubes.

    TMR-dextran Working concentration = 0.05 mg/mL in medium
    2 coverslips at 50 µL/coverslip (=~110 µL/group)

    Add 1751.2 µL medium to 8.8 µL 10mg/mL stock to make 0.05 mg/mL working solution.
    Aliquot ~105 µL/group

    #aliquots 1 2 3 4 5 6 7 8
    vi (10 mg/mL TMR-dex) 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 µL
    medium 218.9 437.8 656.7 875.6 1094.5 1313.4 1532.3 1751.2 µL
    vf 220 440 660 880 1100 1320 1540 1760 µL

    TMR-dextran/ FITC-dextran to stain endosomes in cells in MatTeks
    tmr as above

    FITC-dextran Working concentration = 0.5 mg/mL in medium
    (1mg/mL might be better, but that uses up a whole bottle.)
    2 dishes at 100 µL/dish (=~220 µL/group)

    Add 1742.4 µL medium to an 8.8 µL aliquot of 10 mg/mL TMR-dex plus 8.8 µL 100 mg/mL FITC-Dex

    #aliquots 1 2 3 4 5 6 7 8
    vi (10 mg/mL TMR-dex) 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 µL
    vi (100 mg/mL FITC-dex) 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 µL
    medium 217.8 435.6 650.1 871.2 1089 1306.8 1524.6 1742.4 µL
    vf 220 440 660 880 1100 1320 1540 1760 µL

    Methylamine solution (1M)
    Sigma 426466-100mL 40% w/vol (=12.9M; MW=31) ~$25
    (Working concentration ~20mM - ~20µL/mL. Students need ~100 µL)

    Make 1 mL, aliquot ~120 µL

    Solution stock conc final conc 1 5 10 15 20 mL
    10X PBS 10 1 0.1 0.5 1 1.5 2 mL
    methylamine 12.9 1 78 388 775 1163 1550 µL

    plus water to final volume


    Fixative

    • PBS-paraformaldehyde 3.7%
      • Need 70 mL
      • Divided in 2 aliquots, one per fume hood
    Ingredient 10 15 20 25 40 50 60 70 mL final
    paraformaldehyde 0.37 0.555 2 0.925 1.48 1.85 2.22 2.59 g
    water 9 13.5 18 22.5 36 45 57.78 63 mL
    10X PBS 1 1.5 2 2.5 4 5 6 7 mL

    heat paraformaldehyde in water, then add 10X HBS
    Add a drop of 10N NaOH
    Heat and stir in the fume hood

    OR http://wahoo.nsm.umass.edu/content/paraformaldehyde-fix

    Ingredient 10 15 20 25 40 50 60 70 mL final
    32% paraformaldehyde 1.16 1.73 2.31 2.89 4.63 5.78 6.94 8.09 mL
    10X PBS 1 1.5 2 2.5 4 5 6 7 mL
    water 7.84 11.77 15.69 19.61 31.38 39.22 47.06 54.91 mL

    5.2 2014

    5.2 2014 kdorfman Fri, 10/24/2014 - 19:07

    Cells

    • 3t3 on coverslips - 2 per group
    • 3t3 on MatTek dishes (small diameter)
    • LLCPk GFP-alpha tubulin on coverslips 1 per group
    • LL parentals on coverslips 2 per group

    Reagents

    HBS-BSA

    TMR-dextran to stain endosomes in cells on coverslips
    Invitrogen D3308, 10 mg
    Make 10 mg/mL stock (add 1 mL to the bottle)

    TMR-dextran Working concentration = 0.05 mg/mL in medium

    • 2 coverslips at 50 µL/coverslip (=~110 µL/group) + 2 live cells at 100 µL/mattek
    • Aliquot 5.2 µL 10 mg/mL
    • Label says to add 98.8 µL to make 0.5mg/mL working soloution

    Lysotracker (Invitrogen L-7528 - 20 x 50 µL)

    • 50 - 75 nM
    • stock = 1 mM in DMSO
    • aliquot 10 µL in 2 mL tubes, so they can make 2 mL medium
    • 30 min - 2 hours incubation warm.

    Transferrin in Fe-HBS-BSA

    • Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
    • Each group does 2 50-µL treatments (need extra for pipetting error)
    • Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
    • Aliquot 165 µL

      if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.

    5.2 2015

    5.2 2015 kdorfman Wed, 11/04/2015 - 14:50

    Cells

    • 2 coverslips 3t3 per pair for TMR-dextran
    • 2 LL-GFP-tubulin in MatTek dishes per pair for lysotracker & endosome movement
    • 3t3 in MatTek for live studies

    Reagents

    • HBS-BSA (1 mg/mL). per pair ~7mL:

      • 6 mL for 3 rinses live cells
      • 1 mL for nocodazole
      • 0.1 mL for Transferrin
    • Nocodazole 1 µM in HBS-BSA 1 mL per pair

      • aliquots are 3 µL 3mM in DMSO
      • add 97 µL to make 1mM stock
      • dilute 1:1000 in HBS-BSA (10 µL in 10 mL)
    • Transferrin in Fe-HBS-BSA to label live cells 100 µL/pair

      • Stock is 5 mg/ml (=62.5 µM)
      • Working conc = 1 µM
      • 0.016 µL Tf/µL solution
      • 1 mL = 16 µL Tf + 984 µL Fe-HBS-BSA
    • Lysotracker in non-CO2 medium 1mL/pair

      • stock is 1 mM
      • working solution is 50 - 75 nM
      • Dilute 7.5:100,000 = 0.75 µL/10 mL
    • TMR-dextran (Invitrogen D3308, 10 mg) in non-CO2 medium

      • 100 µL to stain in dish
      • 100 µL to stain 2 coverslips
      • 10 mg/mL stock, in 8.8 ML aliquots
      • 0.05 mg/mL working concentration
      • aliquot ~210 µL per pair
    • CO2 medium (to rinse and return to incubator)

    • Non-CO2 medium (for imaging)

    • PBS for rinsing

    • 3.7% paraformaldehyde in PBS for fixation: 3 mL/pair

    5.3 2013

    5.3 2013 kdorfman Thu, 09/26/2013 - 14:47

    W 10/30

    Transport of endosomes along microtubules

    3 small-diameter MatTek dishes of LLCPk GFP tubulin

    HBS-BSA 1mg/mL

    • to mix transferrin ( 3 mL) and nocodazole (15 mL) in

    • to rinse growth medium off before and after treatment. 6 mL per dish x 3 dishes per group x 8 groups = 144 mL. Aliquot 20 mL.

    • make 250 mL

    To make mL HBS-BSA
    add: mL 10X HBS
    add: g BSA
    plus water to final volume


    Fe-HBS-BSA
    to mix transferrin in
    Need 2.3 mL; make 3 mL

    Solution stock conc final conc 1 2 2.5 3 4 mL
    Fe-citrate (mM) 89.4 0.1 1.118 2.237 2.796 3.35 4.47 µL

    in Fe-HBS-BSA to final volume


    MAKE ENOUGH OF EACH FOR EACH GROUP TO DO 3 TREATMENTS

    Transferrin in Fe-HBS-BSA
    Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD)
    stock is 5 mg/mL (=62.5 µM).
    Final concentration = 1 µM.

    • Each group may do up to 3 100 µL treatments (= 3 x 7 x 105 µL = 2.205 mL)
    • Make 105 µL aliquots (21)
    • Make 2.3 mL: 36.8 µL AF-488-Tf + 2.263 mL Fe-HBS-BSA

    if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.

    # aliquots 1 2 3 6 9 12 15 18 21
    vi 62.5µM tf 1.68 3.36 5.04 10.1 15.1 20.2 25.2 30.2 35.3 µL
    HBS-Fe-BSA 103.3 206.6 310 620 930 1240 1550 1860 2170 µL
    Vf 1 µM tf 105 210 315 630 945 1260 1575 1890 2205 µL

    Nocodazole
    Acros 358240100, 10 mg
    1µM in HBS-BSA
    (Working concentration range is usually 100nM - 30 µM.)
    Stock is 10 mg/mL (=33mM)
    (Add 1mL DMSO to 10 mg in bottle = 33mM. Aliquot 3.03 µL. To make 1mM, add 97 µL.)

    Make enough for each group to do two 1 mL treatments.
    Aliquot 1.01 mL per tube, 15 tubes = 15 mL. Make 16 mL

    • Add 97 µL HBS-BSA to 3 µL 33mM in tube to make 1 mM
    • Mix 1 part 1mM with 999 parts HBS-BSA to make 1µM: 16 mL + 1.6 µL
    # aliquots 1 2 3 4 5 10 15
    vi 1 mM noc 1.01 2.02 3.03 4.04 5.05 10.1 15.15 µL
    HBS-BSA 1.009 2.018 3.027 4.036 5.045 10.09 15.135 mL
    vf 1 µM noc 1.01 2.02 3.03 4.04 5.05 10.1 15.15 mL

    non-CO2 medium
    to mix TMR-dextran in
    to incubate cells in


    TMR-dextran

    in non-CO2 medium
    Each group may do up to 3 treatments = 3 x 7 x 100 µL = 2.205 mL.

    Invitrogen D3308, 10 mg
    Make 100 mg/mL stock (add 100µL to the bottle)
    1 µL aliquots

    TMR-dextran Working concentration = 0.05 mg/mL in medium
    100 µL per treatment
    Aliquot 105 µL, make enough for each group to do 3

    Make 10 mg/mL dilution to make student aliquots
    1 µL 100 mg/mL stock + 9µL medium

    no. aliquots 1 2 3 6 9 12 15 18 21
    vi (10 mg/mL TMR-dex) 0.525 1.05 1.575 2.2 3.15 4.2 6.3 7.875 11.025 µL
    medium 104.5 128.9 656.7 875.6 1094.5 1313.4 1532.3 1751.2 µL
    vf (0.05 mg/mL TMR-dex) 105 210 315 630 945 1260 1575 1890 2205 µL

    6.2 2013

    6.2 2013 kdorfman Thu, 09/26/2013 - 14:49

    W 11/6

    Release of internal calcium stores

    3t3 on large-diameter MatTek dishes

    4 dishes per group (plus extra) ~36

    ATP

    ATP kdorfman Mon, 11/04/2013 - 17:06

    10 µM (=5.731 mg/L) ATP in HBS

    Use ATP aliquots from Lab 6.1


    10 µM ATP, Calcium-free

    Lab 6.2: 0.75 mL per coverslip x 2 coverslips x 8 groups = 12 mL

    Make 15 mL

    • 15 µL 10 mM ATP stock
    • in 15 mL Ca- HBS (1 µL 10 mM ATP stock per 1 mL HBS.)

    8 1.6 mL aliquots for Lab 6.2

    Bradykinin for 6.2

    Bradykinin for 6.2 kdorfman Mon, 11/04/2013 - 17:19

    1mg/mL Bradykinin stock
    (= 1.0602 mM) Sigma B3259 - 1 mg septum bottle
    MW = 106.02
    Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)


    2 µM Bradykinin in HBS

    Use bradykinin aliquots from Lab 6.1


    2 µM Bradykinin, Calcium-free
    (=2.12 µg/mL)

    Lab 6.2: 0.75 mL per coverslip x 2 coverslips x 8 groups = 12 mL

    ~2 µL 1 mg/mL stock per 1 mL working solution

    31.8 µL 1 mg/mL stock in 15 mL Ca-free HBS

    Make 8 aliquots of 1.6 mL

    Make more if necessary. (Aliquots should have been enough for 15 mL?)

    Fluo-4-AM

    Fluo-4-AM kdorfman Mon, 11/04/2013 - 17:20

    Use aliquots from Lab 6.1

    HBS for 6.2

    HBS for 6.2 kdorfman Mon, 11/04/2013 - 17:21

    HBS needed for rinsing and to make other solutions

    (3 rinses + 1 sham dilution) x 4 expts/grp x 1 mL = 16 mL

    Aliquot 25 mL per group

    300 mL HBS

    300 mL Ca-free HBS

    Ionomycin for 6.2

    Ionomycin for 6.2 kdorfman Mon, 11/04/2013 - 17:24

    Ionomycin stock

    Fisher or Invitrogen (Life Technologies) I24222

    1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.

    3 µM Ionomycin in HBS
    Dilute stock 1:333 in HBS: 3 µL/1 mL

    24 µL in 8 mL, aliquot 800 µL

    They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.

    3 µM Ionomycin in Ca-free HBS
    Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
    45 µL in 15 mL

    Takes a long time to thaw. Vortex.

    Aliquot 1.55 mL so they can do two 750 µL experiments

    Vasopressin for 6.2

    Vasopressin for 6.2 kdorfman Mon, 11/04/2013 - 17:26

    1 mg/mL (= 0.9225 mM) Vasopressin stock in water


    1 µM dilution to make student solutions

    1 µL 1 mg/mL in 922.5 µL water (NO CALCIUM, so stock can be used for Ca-free solutions )


    10 nM (= 1.084 µg/mL) Vasopressin in HBS for students

    Use Vasopressin aliquots from Lab 6.1


    Calcium Free Vasopressin 10 nM

    10 nM (= 1.084 µg/mL) for students

    1 part 1µM dilution to 99 parts Ca-free HBS

    Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 8 groups = 12 mL

    Make 15 mL: 150 µL + 15 mL Ca-free HBS

    Make 8 aliquots of 1.6 mL

    Projects 2013

    Projects 2013 kdorfman Thu, 09/26/2013 - 14:50

    starting W 11/13

    Projects

    11/15/13

    11/15/13 kdorfman Wed, 11/13/2013 - 20:14

    Super Group

    • 3 MatTeks parentals 20 mm coverslip
    • non-CO2 medium

    11/18/13

    11/18/13 kdorfman Wed, 11/13/2013 - 19:51

    RA & ML

    • 3 MatTeks 3t3
    • ATP
    • Caffeine
    • Fluo4-AM

    Joon

    • 2 coverslips 3t3
    • 2 GFP-tubulin LL MatTek
    • anti-tubulin
    • paraglut

    On the LAM

    • 4 MatTeks LL GFP actin
    • make serum-free for Tuesday lab

    AV CW AS

    • 5 coverslips LLGFP tubulin
    • 1 matTek alpha tub
    • non-CO2 medium
    • paraglut
    • anti-tubulin

    Super Group

    • 3 MatTeks LL parentals (heavy)
    • 3 MatTek GFP tubulin (heavy)
    • non-CO2 medium
    • Nuc Blue

    11/20/13

    11/20/13 kdorfman Wed, 11/13/2013 - 19:56

    On the LAM

    • 6 MatTek LL GFP-actin HEAVY
    • no serum medium (from night before)

    Dan

    • 3 MatTeks LLCPk tubulin
    • mitotracker

    Super Group

    • 6 Matteks LL tubulin HEAVY
    • mitotracker
    • nuc blue

    RAML

    • 3 Mattek LL parentals
    • Fluo-4

    Cool Team

    • 2 mattek LL tubulin
    • 2 coverslips LL tub

    Joon

    4 MatTek LL tubulin

    11/22/13

    11/22/13 kdorfman Wed, 11/20/2013 - 21:28

    Friday

    Cool Team

    LL = tubulin

    • 1 coverslip
    • 2 dishes

    11/23/13

    11/23/13 kdorfman Wed, 11/20/2013 - 21:28

    Saturday

    Cool Team

    4 dishes LL tubulin

    11/25/13

    11/25/13 kdorfman Wed, 11/13/2013 - 19:58

    Monday

    RAML

    6 dishes LL parentals


    Joon

    4 dishes LL tubulin


    Cool Team

    2 LL tubulin dishes


    Dan

    4 LL tubulin dishes


    On the Lam

    3 LL actin heavy dishes


    Super Group

    4 matteks GFP tubulin HEAVY

    11/27/13

    11/27/13 kdorfman Wed, 11/13/2013 - 19:59

    Cool Team

    3 dishes, 4 coverslips tubulin

    12/2/13

    12/2/13 kdorfman Wed, 11/13/2013 - 20:00

    On the LAM

    • 4 MatTeks LL GFP-actin heavy

    Dan * 4 mattek llcpk GFP actin

    Joon

    • 4 mattek llcpk GFP tub

    Cool team

    • 3 mattek llcpk GFP tub *4 CVslip GFP tub

    Super group

    • 5 mattek GFP tub Heavy

    RAML

    • 4 mattek parental

    12/4/13

    12/4/13 kdorfman Wed, 11/13/2013 - 20:14

    12/6/13

    12/6/13 kdorfman Wed, 12/04/2013 - 21:41

    Super Group

    4 heavy LL tubulin MatTeks


    Cool Team

    2 MatTek LL tub

    1 coverslip LL tub

    12/8/13

    12/8/13 kdorfman Thu, 12/05/2013 - 15:18

    RAML

    4 LL parentals, in dishes

    4 3t3, in dishes

    Microscopes

    Microscopes kdorfman Mon, 01/09/2012 - 15:20

    Nikon Filter Cubes

    label on filter wheel excitation wavelength emission wavelength used for label on cube
    UV UV (360 nm) blue (460 nm) DAPI UV-2E-C
    B B (480 nm) green (535 nm) GFP, fluorescein B-2E/C
    G G ( 560 nm) red (630 nm) Texas red, rhodamine Y-2E/C
    LP B (470 nm) long pass green - red (>500 nm) chlorophyll & GFP simultaneously B-2A

    Details:

    Ultraviolet Excitation Filter Block UV-2E/C Specifications:

    Excitation Filter Wavelengths: 340-380 nanometers (bandpass, 360 CWL)
    
    Dichromatic Mirror Cut-on Wavelength: 400 nanometers (longpass, LP)
    
    Barrier Filter Wavelengths: 435-485 nanometers (bandpass, 460 CWL)
    

    Blue Excitation Filter Block B-2E/C Specifications:

    Excitation Filter Wavelengths: 465-495 nanometers (bandpass, 480 CWL)
    
    Dichromatic Mirror Cut-on Wavelength: 505 nanometers (longpass, LP)
    
    Barrier Filter Wavelengths: 515-555 nanometers (bandpass, 535 CWL)
    

    Yellow Excitation Filter Block Y-2E/C Specifications:

    Excitation Filter Wavelengths: 540-580 nanometers (bandpass, 560 CWL)
    
    Dichromatic Mirror Cut-on Wavelength: 595 nanometers (longpass, LP)
    
    Barrier Filter Wavelengths: 600-660 nanometers (bandpass, 630 CWL)
    

    Blue Excitation Filter Block B-2A Specifications:

    Excitation Filter Wavelengths: 450-490 nanometers (bandpass, 470 CWL)
    
    Dichromatic Mirror Cut-on Wavelength: 500 nanometers (longpass, LP)
    
    Barrier Filter Wavelengths: 515 nanometer cut-on (longpass, LP)
    

    DIC

    DIC bcrcstaff Tue, 11/20/2018 - 15:29

    (On microscope 7)

    Prism combinations

    objective magnification prism on condenser turret prism on objective turret
    Plan Fluor phase 10x N1 10x
    Plan Fluor phase 40x (short working distance) N2 40x I
    S Plan Fluor phase 40x (extra-long WD) N2 40x III
    Plan Fluor phase 100x oil N2 100x II
    Achromat w/iris brightfield 100x oil no DIC

    Posters

    Posters kdorfman Wed, 12/03/2014 - 18:42

    Supplies

    Supplies kdorfman Thu, 11/03/2011 - 19:35

    Get coverslip-bottom dishes from Cellvis, much cheaper than MatTek.

    60 mm

    35 mm

    29 mm

    Celltreat also has them, about $2/dish:

    https://www.celltreat.com/30mm-x-10mm-tissue-culture-treated-dish-15mm-…


    Microscope bulbs for phase: 12V 100 W

    • Philips 77241 or
    • Osram HLX 64623

    http://www.planetbulb.com/osram-eva-64623-hlx-54251/

    Microscope bulbs for Zeiss:

    • 38-01-77 6V 15W
    • OQ-77Z 3800-18-1740 6V 15W or
    • WW-6W51-8 from interlight.biz

    Microscope bulbs for Olympus: BLC 120V 30W S11 BA15D from interlight.biz


    Nucleofection kit

    Ingenio Electroporation kit, MIR50112

    www.mirusbio.com


    mounting medium (SouthernBiotech Dapi Fluoromount-G, Fisher OB010020)

    Hoechst solution Invitrogen™ Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water Nucleic acid stain Invitrogen™ H3570 cheaper than Thermo Fisher