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

reticule1.jpg

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

ceramide.pdf

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

(E. coli for plasmids are in -80)

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
Promega midiprep manual
Addgene plasmid concs

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

er-tracker.pdf

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

t110.pdf

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

blebbistatin-info.pdf

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
ertracker.pdf