5-methylcytosine (D3S2Z)

Rabbit mAb

Binds to methylated cytosine

28692S Cell Signaling Technology

From the manufacturer: "Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1

Dilute 1:1600

Before Antibody treatment, treat fixed cells with 4N HCl for 15 minutes to denature DNA.

Neutralize with 2 washes with sodium borate pH 9, then PBS. Then start the PBS-Tw-Az rinses before incubating with the antibody.

Store at -20

DSHB JLA20

stock concentration = 22 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 90 uL ~11 mg/mL

Add 216 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

Sigma A2668

Dilute 1:40 for use

Dilute 1:1 with glycerol

Aliquot 16 uL

Add 290 uL PBS BSA to make enough for 3 slides

Freezer 266A

DSHB B4-78

stock concentration = 38 ug/mL

diluted 1:1 in glycerol

50 uL aliquots of ~19ug/mL

Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides

266A freezer

Binds actin to membrane at adherin junctions
Mouse monoclonal, Clone BM 75.2
IgM
Fix in formaldehyde (no MeOH or glutaraldehyde)
1:200
Use anti-mouse IgM FITC secondary

"calcium-dependent adhesion"

cell adhesion molecule (CAM) important in formation of adherens junctions to bind cells with each other.

Class of type-1 transmembrane proteins.

Dependent on calcium (Ca2+) ions to function

small aliquot: 5 µL
large aliquot: 10 µL

DSHB II-II6B3

stock solution: 96 ug/mL

working solution: 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 20 uL ~48 mg/mL

Add 286 uL PBS-Tw-BSA to make enough for 3 slides

266A Freezer

DSHB M3F7

mouse anti collagen IV (basement membranes)

2 tubes as of spring 2018:

• stock concentration (8/7/08) = 30 ug/mL
• stock concentration (12/22/11) = 39 ug/mL

working concentration = 2 - 5 ug/mL

Each diluted with glycerol to a concentration of 18.5 ug/mL

Aliquots are 50 uL

Add 256 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

Freezer 266A

Anti-connexin-32 (265-279)

Sigma C3470

Dilute 1:600

Anti-Connexin-32 (106-124)

Sigma C3595

Dilute 1:400

DSHB R5.21C

stock concentration = 28 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 70 uL ~14 mg/mL

Add 236 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

D63A6 XP(R)

Rabbit mAb

5032S Cell Signalling Technology

detects endogenous levels of total DNMT1 protein.

From the manufacturer: "Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2 and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication."

Dilute 1:100 for use

1 µL aliquots in tube. Store in freezer (-20C)

add 100 µL 1% BSA in PBS

Use just under 50 µL per coverslip (so there is enough for 2 per tube)

[edited 9/26/18 - used to say 1:200 dilution, but we had better results with 1:100]

Novus Biologicals, recombinant human Hec1, monocolonal
fix in methanol
dilute 1:200

FINISH WORKING ON THIS!

Rabbit polyclonal

Abcam AB5176

1 mg/mL, 100 ug, should be 100 uL in the package

1:2000

Storage:

• Dilute 1;10 glycerol
• 2 uL aliquots; for use, raise to 400 uL
• leave the rest with a label: dilute 1 uL in 200 uL PBS-Tw-Az-BSA

DSHB Ralph 3.1

rat anti-integrin-3

IgG2a

stock concentration = 27 ug/mL

working concentration = 2 - 5 ug/mL

Freezer 266A

For Gene & Genome (Maresca)

From Wikipedia Antigen KI-67 is a nuclear protein that is associated with cellular proliferation and ribosomal RNA transcription. Inactivation of antigen KI-67 leads to inhibition of ribosomal RNA synthesis, but does not significantly affect cell proliferation in vivo: Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently

Fix cells in 4% PFA

1:500 rabbit. worked great 11/21/22

GeneTex GTX13523

IgG

1 mg/mL

4C short term

Freeze 1 µL aliquots

dilute 1:100 - 1:750 in 0.1% BSA in PBS

rinse in PBS

Fix

• in 4% paraformaldehyde 15 min for endosomes
• in cold methanol for Golgi, 0.5% saponin 5 min

1% BSA in PBS 30 min

1o Ab stain (0.1% BSA) overnight at 4C
wash 30 min in PBS

2o Ab 1 h 37C
wash 30 min PBS

mount in DAPI

reference

Stains nuclear envelope

• fix in cold methanol
• 1:100
• BD transduction
• Mouse IgG

Cell Signalling 4777S

mouse monocolonal (4C11)

7 ug/mL

Use at 1:100

(20 uL/class)

Storage:

• dilute 1:1 with glycerol,
• make 5 uL aliquots;
• add 245 uL PBS-Tw-Az-2% BSA

Rabbit anti-Lamin B1

Abcam AB16048

comes 100 µL at 1 mg/mL

Says to use at 0.1 µg/mL = 1:10,000 Drew's lab uses it at 1:1000

Make aliquots:

• 2 µL in a 2 mL tube
• label says
  • 2 µL rabbit anti-lamin B1
  • add 2 mL PBS-Tw-Azide

Aliquot and freeze - do not refreeze

DSHB 2E8

mouse anti-laminin gamma 1

stock concentration = 26 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 80 uL ~12 mg/mL

Add 226 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

https://www.sigmaaldrich.com/catalog/product/sigma/l9393?lang=en&region…

Sigma L9393

3 vials as of Spring 2018

dilute 1:25
266A freezer

Monoclonal Anti-Mucin Gastric antibody produced in mouse

Sigma M5293

dilute 1:200 for use

dilute 1:1 in glycerol

Aliquot 4 uL; add 302 uL PBS BSA to make enough for 3 slides

Freezer 266A

DSHB T14

mouse anti-myosin light chain

stock solution: 37 ug/mL

working solution: 2 - 5 ug/mL

diluted 1:1 in glycerol

50 uL aliquots of ~18ug/mL

Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides

Freezer 266A

Sigma M7523

Formalin fixation

2 vials as of spring 2018

Dilute 1:20

freezer 266A

Anti-Neurofilament 200 antibody produced in rabbit

Sigma N4142

Dilute 1:80

Freezer 266A

abcam ab24609

Data Sheet

Anti-Nuclear Pore Complex Proteins antibody (Mab414)

100 µL, 1 mg/mL

Dilute 1:1000 to use

2 µL aliquots. Label says:

• add 198 µL PBS-Tw-Az-BSA
• dilute again 1:10 for use

Mouse monoclonal [Mab414] to nuclear pore complex proteins

Suitable for ICC or IF

Reacts with mouse, human, Saccharomyces cerevesiae

Isotype: IgG

Store at 4C short term

Aliquot and freeze for long term storage

Fix in 100% methanol -20C OR 4% PFA in PBS pH 7.4 10 min RT

PKC alpha (c-20)

rabbit polyclonal Abs 1gG

DO NOT FREEZE

In refrigerator 36sA

dilute 1:50 to use

Santa Cruz Biotechnology sc-208

From Wikipedia: Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control. Knockout studies in mice suggest that this kinase may be a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes.[5]

Protein kinase C-alpha (PKC-α) is a specific member of the protein kinase family. These enzymes are characterized by their ability to add a phosphate group to other proteins, thus changing their function. PKC-α has been widely studied in the tissues of many organisms including drosophila, xenopus, cow, dog, chicken, human, monkey, mouse, pig, and rabbit. Many studies are currently being conducted investigating the structure, function, and regulation of this enzyme. The most recent investigations concerning this enzyme include its general regulation, hepatic function, and cardiac function.

For showing mitotic cells

Histone H3 phosphorylation on serine-10 is specific to mitosis and phosphorylated histone H3 (PHH3) proliferation markers (as counts defined per area or as indices defined per cell numbers) are increasingly being used to evaluate proliferation in various tumors.

Dilute 1:2000

link

DSHB SV2

Synaptic vesicle glycoprotein 2A

Stock concentration = 36 ug/mL

Working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 60 uL ~18 mg/mL

Add 246 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

DSHB TI-4-s

Anti-troponin I

IgG (mouse)

Stock concentration = 43 ug/mL

Working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 50 uL ~22 mg/mL

Add 256 uL PBS-Tw-BSA to make enough for 3 slides

266A Freezer

Accurate Chem YSRTmcADg YSRTMCA77G

Clone YL 1/2

Antibody as purchased (2019) was diluted 1:1 with glycerol

As purchased, should be diluted 1:200; glycerol-diluted AB should be diluted 1:100 with PBS-Tw-Az-BSA

Each tube has 4 µL antibody

Add:
400 µL PBS-Tw-Az-BSA =404 µL, enough for 8 reactions (50 µL per coverslip, 60 min 37C)

Incubate coverslip on 50 uL antibody 1 hr 37C in humid chamber

binds to centrosomes

From Pat
Monoclonal, Sigma clone GTU-88
Fix in paraformaldehyde or cold methanol
Dilute 1:100

From Drew Sigma T6557-100UL

• Working concentration is 1:5000
• Frozen stock is diluted 1:1 with glycerol
• 5 uL aliquots (in half-mL tubes) should be diluted:
  • 1:50 in the tube: add 245 uL PBS-Tw-BSA to make 1:100 stock
  • 1:50 for use (1 uL 1:100 antibody + 49 uL PBS-Tw-Az-BSA per coverslip)

Drew's Protocol:

• Rinse in PBS
• Fix in paraformaldehyde 15 minutes
• Rinse with PBS-Triton
• Rinse with PBS-Tween
• Rinse with PBS-BSA 1 hour
• Dilute Ab 1:5000 in BSA
• Incubate with Ab 2hr RT
• Rinse with PBS 3x
• Incubate with secondary Ab (in BSA)
• Rinse with PBS 3x
• Mount

Test Protocol:

• coverslips already fixed in paraglut, stored in PBS-Tw-Az ¹
• 1 uL Ab (as supplied by Sigma) into 99 uL PBS-BSA (save!)
• 1 uL 1:100 Ab into 499 uL PBS-BSA (save for the rest of the slides if it works!)
• 50 uL onto coverslip - 2 hr RT incubation
• rinse with PBS-Tw-Az
• 50 uL Goat anti mouse 488

Membrane-cytoskeletal protein in focal adhesion plaques that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton, associated with cell-cell and cell-matrix junctions, where it is thought to function as one of several interacting proteins involved in anchoring F-actin to the membrane.

Sigma V4505 monoclonal mouse
Fix in Formaldehyde
Dilute 1:100

large tube has 10 µL antibody

small tube has 5 µL antibody

use at 1:100

362A freezer

fix in paraformaldehyde

Fisher OB102102 (Southern BioTech 1021-02)

Goat Anti-Mouse IgM (micron chain specific)-TRITC

1 mg/mL

1:100 - 1:400

1 µL aliquots. Add 100 - 400 µL 1% BSA

Use 50 µL per coverslip

Invitrogen A-11001

Ex: 488 nm

Em: 499 nm

Stock(as purchased) = 2 mg/mL

Stock bottle has been diluted with glycerol: ~800 uL of 1 mg/mL

Working concentration = 1 ug/mL

Aliquots are 1 uL of 1 mg/mL

Add 611 uL to make enough for 6 slides, or up to 1 mL

Freezer 266A

Anti-Mouse IgG (whole molecule) TRITC conjugate

Sigma T 5393

26 mg protein/mL

minimum working dilution = 1:64

1 µL aliquots. Add up to 100 µL 1% BSA

Alexa Fluor 568 (A11004) (red) 2mg/mL

working concentration ~10µg/mL
dilute 1:200

aliquots are 110 µL 20µg/mL

add
110 µL 2% BSA in PBS
to make 220 µL 10µg/mL

Use 50 µL per coverslip (there is enough for 4 per tube)

Molecular Probes A-11010

Stock concentration = 2 mg/mL

Working concentration = 4 ug/mL

Em = 575 nm

Ex = 560 nm

ALL OUT as of Spring 2018

Goat anti-rabbit IgG FITC

Sigma F0382

Dilute 1:80

Jackson Immuno 111-165-003

Cy3 Ex - 550 nm, em= 570 nm ("Red")

tubes : 2 µL. Add 398 µL PBS-Tw-Az to make 1:200

Use 50 µL per coverslip (there is enough for 4 per tube)

Sigma F6258

Each tube has 3 µL antibody

Add
192 µL PBS-Tw-Az
195 µL 2% BSA
=390 µL, enough for 7 reactions (50 µL/coverslip 37C 30min)

From Sigma AP136R EMB Millipore

ab peak = 550nm; emission peak = 570 nm

Bottle contains 2 mg. Add 1 mL H2O + 1 mL glycerol to make 1 mg/mL

aliquot 2 µL or 5 µL antibody, freeze

add 198 µL or 495 µL PBS-Tw-BSA

use 50 µL per coverslip, diluted to 1/100 (Try 37C 30 min)

10X HBS/Ca/Mg from dry ingredients

pH 7.3 with NaOH

Filter sterilize

10 X HBS from stock solutions

pH 7.3 with NaOH

Filter sterilize

pH 7.3 with NaOH

Filter sterilize

Alkaline Lysis Reagent for HotSHOT DNA prep

Recipe from Mike Charles 10/15/03; mikchar@umich.edu; retrieved from Craig Albertson's lab, recalculated for1 M NaOH - 10 M will damage your plastic tube!!

Calculator

pH =~12

Neutralization Buffer for HotSHOT DNA prep

Dilute stock Tris-HCl to 40 mM

pH =~5

10X PBS from stock solutions

from Sigma ready-made 1X:

pH to 7.3 with NaOH

sterilize

OR

To match Sigma recipe:

Make 1M Na2HPO4 (base); 1 M KH2PO4 (acid)

Mix to pH 7.3

(should be ~38.25 mL Na2HPO4, 11.5 mL KH2PO4) from the Sigma Buffer Reference Center

Then

plus water to final volume

Buy from Fisher TAE 50x, pH 8.3, 4L, part #BP13324

from Maniotis

pH to 8 with acetic acid or NaOH

should this be 8.4?

pH to 8 with acetic acid or NaOH

Maniotis says to autoclave, but the salt concentration is so high that nothing will grow in it if you don't.

To Freeze:

• Pick a new colony, grow overnight in LB

• inoculate LB from the overnight culture

• Put 0.15 mL glycerol into sterile cryovial.

• Add 0.85 mL mid-log culture (OD =~0.4). Pipette up and down to mix thoroughly

• Freeze. Store in -80 if possible.

To thaw:

• Scrape surface of frozen stock with sterile stick

• Streak on agar plate

Table of antibiotic stability in poured agar

LB + sugars for Lac-operon work

Filter sterilize

Add 50 µg/mL Kanamycin as indicated here.

MacConkey Agar

• Fisher 212122 2 kg (Difco)
• Krackeler 10-211387 500g (via Sigma)
• HiMedia MacConkey agar, granulated GM081 (Fisher NC1876763, VWR 95020-204)

to distinguish Lac+ and Lac- bacterial strains.

50 g/L

(If you are going to make more than one bottle, turn on 65C waterbath in tissue culture room)

Turn on the stir-plate heat to 105C.

Withhold 50 mL of water to rinse the mouth of the bottle.

Heat the rest of the water to boiling in the bottle in the microwave.

Measure out the agar mix while the water is heating

Add the stir bar (CAREFULLY!) to the bottle of hot water

Put the bottle on the stir-plate

Add the agar mix to the bottle

Use the reserved water to rinse the granules stuck in the neck down to the liquid.

Stir and heat until granules are completely dissolved.

Cap loosely, wrap with foil, and autoclave.

(If you make more than one bottle, the extras go in the water bath till you are ready to pour)

leave stir bar in

put bottles on stir plate near sterile hood until handle-able

If you accidentally buy

From https://wahoo.cns.umass.edu/node/857

For SfGFP cells, try M9 + 0.2% glycerol + 0.01 mM FeSO4

90922 Mueller Hinton Broth 2 from Sigma

22 grams per liter, consisting of:

Casein acid hydrolysate 17.5
Beef extract 3.0
Starch 1.5

Final pH 7.3 +/- 0.2 at 25°C

Make 60 mm plates

• plain LB plates

• LB/Amp (or Carb) plates (1 black stripe)

• LB/Amp/Ara plates (1 black, 1 red stripe)

2019 - for QBoC:

• transformed pglo plasmid (1660405EDU) from the BioRad kit into
• DH5 alpha E. coli (Invitrogen 18265-017) from the -80
• Lyophilized cells (BioRad 1660408EDU) did not arrive in time

Used the Invitrogen protocol.

Plated on:

• LB
• LB amp
• LB amp ara

Saw gfp colonies on the arabinose plates. Collected, grew up in LB amp, froze in 15% glycerol.

For motility strains

FBS comes in 500 mL bottles.

Aliquot:

2013: Try

• Fisherbrand™ Research Grade Fetal Bovine Serum
• 03-600-511
• 500 mL for $133

Krackeler 45-F0926-500ML $145

Carrie's brand: Atlanta Biologicals Premium S11150 ~$300

Also use Krackeler 12103C ~$314

2019 Genessee GenClone FetalPURE™ Cat #: 25-525 $247.45

Anti-Anti comes in 100 mL bottles.

Aliquot:

Keep frozen until ready to use.

Fisher SV30079.01, $20

from Kathleen Arcaro

bring to final volume, filter sterilize in BSC, refrigerate

insulin solution 10 mg/mL in HEPES

amino acid solution

Medium for 3t3 fibroblasts and B16 cells

bring to final volume, filter sterilize in BSC, refrigerate

• if using DMEM without pyruvate, add 1 mL Na Pyruvate (100mM) per 100 mL medium

Mix DMEM, NaHCO3, HEPES in about 70% of the final volume of dH2O.
Initial pH ~7.5
Adjust pH to 7.2 with HCl (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.

for all LLCPk cell lines

Thaw FBS and anti-anti first!

bring to final volume, filter sterilize in BSC, refrigerate

Mix F10, Optimem, NaHCO3, HEPES in about 70% of the final volume of dH2O.

Initial pH ~7.1
Adjust pH to 7.2 (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Refrigerate

For serum free, replace serum with distilled water

Medium with 15% DMSO and 20% serum to protect cells in liquid nitrogen.

F10 Hams for freezing (initial serum concentration = 0.075)

DMEM for freezing (initial serum concentration = 0.1)

Filter sterilize

McCoy's 5A (modified) (Fisher 16600-082)

For HCT116 cells (a human colorectal carcinoma cell line initiated from an adult male. The cells are adherent with an epithelial morphology. Following implantation into immunocompromised mice, the cells form primary tumors and distant metastases. In vitro, HCT116 cells grow with a doubling time of about 18 hours.)

Drew's recipe:

10% FBS, 1% Pen-strep

• 500 mL McCoy
• 55 mL FBS
• 5.5 mL pen-strep

OR:

• remove 55 mL from the 500 mL McCoy's from Fisher
• add 50 mL FBS
• add 5 mL pen strep (e.g., MP 1670246)
• resterilize

Filter sterilize

Try this: http://www.thermofisher.com/us/en/home/life-science/cell-culture/mammal…

FluoroBrite™ DMEM Media

Try Fisher 12-605-010

Stable at room temp!

Gibco™ TrypLE Express Enzyme (1X), Phenol Red

Animal origin-free, recombinant enzyme

$20.10 -for 100 mL

1 gram of methylene blue per liter of RO water.

Shake well to dissolve.

Store at room temp.

FOLLOW THE RECIPE IN THE ATTACHED FILE (omitting methylene blue). THIS PAGE IS NOT DONE YET

100X E2:

1.5 M NaCl

50 mM KCl

100 mM MgSO4

15 mM KH2PO4

5 mM Na2HPO4

Mark with 2 red lines

Add 1 black line for carbenicillin (ampicillin)

Add 1 green line for IPTG

If required, add IPTG 0.5mM (0.5 mL 1M per L medium) to 1 mM (1 mL 1M per L medium),

and ampicillin

DIW (pure deionized water) 780 ml

1M K2HPO4 (dibasic) 4 mL

1M KH2PO4 (monobasic) 16 mL

Difco Bacto Peptone (not Proteose Peptone) 3.2 g

Difco Bacto Agar 12.0 g

Mix by swirling (no need to dissolve). Autoclave 10 min. Cool to 45–50° in a water bath, and pour plates about ½ full.

1/2 Murashige & Skoog, 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM, 1/2 MS is 5µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

C Fern Manual

Carolina C-Fern materials

Pre-made fern medium, Carolina 156780, sold in 60 mL or 400 mL

Carolina recommends 10 mL per plate. I use 9 mL

To get gametophytes in all stages, sow fern spores in 12 60 mm dishes 4 times:

• 1/15
• 1/22
• 1/29
• 2/2 or Friday 2/3

Lilly pollen growth medium

205 mM (7%) sucrose (See Sugars.)

1.6 mM H3BO3

0.1 mM CaCl2

15 mM MES

Either make fresh each day, or filter sterilize for all the labs.

Stock solutions:

150 mM MES

10 mM CaCl2

160 mM Borate

150 too high - try 100mM for F 2015

150 mM NaCl

10 g agar/L

Make regular MS, then add 0.03 mL 5M NaCl per mL medium

100 mM NaCl

0.02 mL 5M NaCl per mL medium

Murashige & Skoog 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

10 g agar/L

Murashige & Skoog 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

Wikipedia entry

Before you start:

• check for 20% glucose, filter sterilized; make more if needed
• warm the glucose (a ~60C oven is best; use a 37 incubator if necessary)
• sterilize a graduated cylinder if volume of glucose to be added is more than 50 mL

per L:

• 40 g glucose
• 10 g peptone
• 20 g agar
• pH 5.6

Per 1 L

• 100 mL 10X M9 salts
• 240 mL glycerol
• 300 µL 1M MgSO4
• water to 1L

Filter sterilize

Per 1 L:

• 100 mL 10X M salts
• 300 µL 1M MgSO4
• water to 1 L

Filter sterilize

Per Liter of medium (~75 plates):

• 975 mL Water
• 3 g NaCl
• 2.5 g Peptone (Fisher BP1420-500 $78.80)
• 17 g Bactoagar

Autoclave with stir bar inside

Cool to 55C in a 55C water bath

Add per L (see recipes in stock solutions):

• 1 mL cholesterol (5 mg/mL in 95% EtOH)
• 1 mL CaCl2 (1 M, STERILE)
• 1 mL MgSO4 (1 M, STERILE)
• 25 mL K-phosphate buffer (1M, pH 6.0, STERILE¹)

Swirl flask to mix

Dispense 10 mL into each 60mm dish.

Stack 10 high

Let stand for ~48 hours for condensation to evaporate

Pack in sterilized plastic boxes.

put bottles on stir plate near sterile hood until handle-able

For Bio 284

• 0.15 g YNB
• 0.52 g ammonium sulfate
• 2.0 g agar
• 82 mL H2O
• 8.0 mL 1 mg/mL adenine
• 10.0 mL 20% glucose (final conc = 2g/100 mL)

Minimal Vitamin Medium plus Adenine

Label plates with "+ ADE"

Make sure it's a plus sign!

Autoclave an appropriately sized graduated cylinder to measure the glucose and adenine solutions.

Mark with two blue lines

*Yeast Nitrogen Base without amino acids and ammonium sulfate

Minimal Vitamin Medium for Bio 284

Autoclave an appropriately sized graduated cylinder to measure the glucose solution.

*Difco Yeast Nitrogen Base w/o Amino Acids and w/o Ammonium Sulfate

Mark with a single blue line

Plates for Jeff Laney's Cell & Molecular Biology Lab

SC-Leu low-Ade plates for Jeff Laney's cell and molecular biology lab

SC minus (adenine, histidine, leucine, tryptophan)

Sunrise Science 1330-030

4X will dissolve. Almost completely clear after overnight stirring. Crystal clear after autoclaving.

From the manufacturer:

Commonly added to YNB, nitrogen and glucose, at suggested g/L, for a complete yeast media. Mix with stirring for 10-15 minutes, and filter sterilize or autoclave at 121°C for 15 minutes. To prepare plates, autoclave agar separately and add to sterile medium, or add agar to liquid medium and adjust to pH 5.8-6.0 before autoclaving.

Suggested g/L: 1.64

Storage Temperature: 2-8 C

Jeff's recipe: 10X SC –AHLT 16.4 g/L

Dissolve 16.4 g of Sunrise Science SC –Adenine, –Histidine, –Leucine and –Tryptophan in 1 L distilled water and filter sterilize.

Store in the dark at 4 ˚C.

Will not go into solution.

Concentrate to make SC-L High Ade or Low Ade agar

Make this concentrate, then add adenine hemi-sulfate.

for low-Ade, add 5 mg Ade per L of concentrate

for high-Ade, add 1.6 g Ade per L of concentrate

to make high-Ade from low-Ade concentrate, add 1.595g Ade per L, or 0.1595g per 100 mL

Then filter sterilize. For every L of medium, combine 20g agar autoclaved in 875 mL water with 125 mL warmed up concentrate.

add 125 mL concentrate to autoclaved 875 mL water + 20 g agar

Liquid Medium

Using 4X SC-AHLT (because 10x won't go into solution):

Bring to final volume with water, then filter sterilize.

Jeff Laney's original (discontinued - because you can't make 10X SC-AHLT) recipe:

Filter sterilize

Store at room temperature

Yeast Extract Adenine Dextrose Medium

Ade mutants grow well on this without turning red. Better for keeping the parental strains white longer.

1 gram Yeast Extract 2 grams anhydrous dextrose (glucose) 2 grams Agar 8 mL adenine stock solution (1 mg/mL)

Adenine Stock Solution

400 mg adenine in 400 ml water (1 mg/ml) Store at room temperature. Use 2 ml stock solution for 100 ml of medium; reduce the water added to the medium by 2 ml.

Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.

Yeast Extract Dextrose

For Bio 284

Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.

NOTE: 900 mL may boil over in a 1 L bottle in the autoclave. Try:

• Autoclave:
  • YE + agar in 800 mL
  • 100 mL H₂O
  • mix in BSC
• Add 100 mL sterile 20% glucose
• mix and pour

Mark with a single red line.

Sporulation Medium for Bio 284

If you have concentrated potassium acetate solution, then:

• 20.4 mL 5M KOAc per liter
• 12.75 mL 8M KOAc per liter Fisher AAJ63372AE

Mark with a single black line.

If you are using potassium acetate powder, then:

YEPAD medium (Yeast Extract + Peptone + Adenine + Dextrose) contains the same ingredients as YEAD but has peptone added. This is a very rich medium used for storing yeast strains.

1 gram Yeast Extract 2 grams Peptone 2 grams anhydrous dextrose (glucose) 2 grams Agar (agar-agar; gum agar) 8 mL adenine stock solution 92 ml water

A Dohlman Lab Protocol

Yeast strains, transformed or untransformed, can be maintained as colonies on solid media at 4° and restreaking every 2 to 4 weeks. Alternatively, strains may be stored at -80 indefinitely. This is preferable in that it reduces the likelihood of accumulating spontaneous mutations.

To make frozen stocks of yeast strains: -Use sterile technique and sterile solutions throughout this method.-

1. Grow a starter culture at 30 with shaking (250 rpm) until it reaches saturation.

2. In a 1.8 ml cryotube, mix 0.5 ml of the saturated culture with 0.5 ml of YPD containing 20% glycerol.

3. Flash freeze the tube in liquid nitrogen and store at -80.

4. To use, chip out a few pieces of the frozen stock using a sterile pipette tip or sterile toothpick and streak onto a plate containing the appropriate solid media.

5. Allow the yeast to grow at 30 until colonies appear (2-6 days).

10X YNB+AS

17 g/L YNB and 50 g/L AS

Dissolve 17 g Difco Yeast Nitrogen Base w/o Amino Acids and w/o Ammonium Sulfate and 50 g Ammonium Sulfate in 1L distilled water.

Autoclave for 15 min.

Store at room temp.

Yeast extract-Peptone-Dextrose medium:

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.

Sigma A9525 1mg/mL in water

Vasoconstrictor

Soluble in water 25 mg/mL (only use sterile water!)

MW: 1046.18 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

Subject to degradation in freezer storage - wear gloves!

Make 1 mg/mL stock (= 1.0462 mM)

Working concentration 1 µM

Dissolve 1:1000 in HBS

BMS 833923

Cayman Chemical 16240

Smoothened (Smo) is a cell surface receptor that, with Patched, mediates sonic hedgehog (Shh) signaling to regulate gene expression through the Gli transcription factors.1 BMS 833923 is an orally bioavailable inhibitor of Smo.2,3 It blocks binding of BODIPY cyclopamine (IC50 = 21 nM) and inhibits Gli activation in cell lines that express wild-type Smo or activated mutant forms of Smo (IC50s = 6-35 nM). BMS 833923 robustly inhibits Shh pathway activity and prevents tumor growth in medulloblastoma and pancreatic carcinoma xenograft models.

References

1. Ruiz-Gómez, A., Molnar, C., Holguín, H., et al. The cell biology of Smo signalling and its relationships with GPCRs. Biochim. Biophys. Acta1768(4), 901-912 (2007).

2. Sandhiya, S., Melvin, G., Kumar, S.S., et al. The dawn of hedgehog inhibitors: Vismodegib. J. Pharmacol. Pharmacother.4(1), 4-7 (2013).

3. Lin, T.L. and Matsui, W. Hedgehog pathway as a drug target: Smoothened inhibitors in development. OncoTargets and Therapy5, 47-58 (2012)

Bisphenol A

Sigma 239658

MW 228.29

100 mM stock in DMSO = 0.023g/mL

Sulfonyldiphenol

Sigma 103039

MW = 250.27

100 mM stock in DMSO = 0.025g/mL

Myosin inhibitor

(-)-Blebbistatin 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

• 9-amino acid peptide chain: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Bradykinin raises internal calcium levels

*Sigma B3259 - 1 mg septum bottle

• MW = 1060.2
  

  1 mg/mL stock
  (= 1.0602 mM)

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)

Fisher 00-4506 1000x solution

Keep in refrigerator

Description

Brefeldin A is an inhibitor of intracellular protein transport. Incubation of cells in culture with Brefeldin A leads to blockade of protein transport to the Golgi complex (GC) and accumulation of proteins in the endoplasmic reticulum (ER). Addition of Brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines. Brefeldin A is effective for enhanced detection of a majority of mouse and human intracellular cytokines; however, it is advised that the investigators evaluate the use and efficacy of this reagent as well as other protein transport inhibitors such as Monensin in their specific assay system.

Applications Reported

The Brefeldin A Solution is supplied at 1000X working concentration in methanol. Prior to addition to the cell cultures, a 1:1000 dilution in the culture media should be made. The final working concentration of the Brefeldin A (at 1x) is 3.0 ug/ml.

MW 194.19

Soluble 10 mg/mL in water (with heat and stirring)

Make a 50mM (=0.05M) solution: 9.71mg/mL

Tetrahydrocannabinol solution 1 mg/mL in methanol

Sigma T464

store at 4C

Vial is in a white box in fridge door, room 362A

PLK4 (Polo-like kinase) inhibitor

Plk4 has been identified as a master regulator of centriole replication, and its aberrant expression is closely associated with cancer development

PLK4 functions downstream of ROCK2 to drive centrosome amplification in arrested cells. PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.

100uM stock

• use 125 nM for HeLa (2.5 uL stock in 2 mL medium)

• use 300 nM for LLCPk-1 (6 uL stock in 2 mL medium)

• Sigma C5275 5 mg
• Plant mitogen
• Lectin (carbohydrate-binding protein) extracted from the jack-bean, Canavalia ensiformis

• Con A binds specifically to α-Mannose, α-Galactose structures found in sugars, glycoproteins and glycolipids

• Make stock 5 mg/mL solution (add 1 mL sterile PBS to bottle – gently rotate to mix)

• Keep sterile - work in sterile hood

• aliquot 100 µL into microfuge tubes  (cannot take repeated freeze-thaw cycles)

CKD1 inhibitor IV RO3306

1 mM in DMSO

A cyclin-dependent kinase inhibitor protein inhibits cyclin-dependent kinase. Several function as tumor suppressor proteins. Cell cycle progression is negatively controlled by cyclin-dependent kinases inhibitors (called CDIs, CKIs or CDKIs). CDIs are involved in cell cycle arrest at the G1 phase.

Protein synthesis inhibitor

Fisher AC357420010

MW = 281.35 g/mol

C15H23NO4

Solubility in water: 2.1 g/100mL (

soluble in chloroform, ether, acetone, methanol, and ethanol

powder

ApexBio A8340

10 mM

Store in freezer

C27H41NO2

MW = 411.62

Soluble 6.86 mg/mL in DMSO (warm to 37C and shake to get it into solution)

Reaction Conditions :

• 20 μM, 48 hours for cell yield inhibition
• 10 μM, 48 hours for apoptosis induction (measured by PARP expression)

Cyclopamine is a naturally occurring Hedgehog (Hh)?specific small?molecule signaling steroidal alkaloid inhibitor, causes a profound inhibition of tumor growth, has significant anti?invasive, anti?proliferative and anti?estrogenic potency in human breast cancer cells [2] [1]. The EC50 of cyclopamine is 10.57 μM, it was identified by an FXR-bla (farnesoid X receptor- b-lactamase) assay [3].

Hh signaling pathway plays a critical role in embryonic development and tumorigenesis [4]. Hh signaling pathway shows saliency in regulating cellular proliferation and differentiation in a wide array of human tissues. It is related to aberrant cell survival in numerous human malignancies, ranging from BCCs and medulloblastomas to small cell lung, gastrointestinal, breast and prostate tumors [1].

Treated with cyclopamine (10 or 20 μM) only and incubated for time periods ranging from 0 to10 days, MCF-7 cells and MDA?MB?231 cells displayed a significant reduction in proliferation rate compared with the control cells on days 3 and 6 (P

Embryoes exposed to cyclopamine resulted in visible external defects, including cyclopia, proboscis formation, microphthalmia, thoracic lordosis, amelia and decreased body size. Examination of gastrointestinal organs revealed severe deficits, including less length of the gut tube and mesenchymal cell numbers in foregut-derived organs. Ectopic structures in duodenum, stomach, and dorsal pancreas were also found [5].

MW = 507.62
Sigma C2618: 200 µL 5 mg/mL (~10 mM) in DMSO
Sigma C8273: 5 mg. Dissolve in 1 mL DMSO

• alkaloid mycotoxin produced by Helminthosporium and other molds.
• disrupts actin microfilaments
• binds to F-actin polymer
• prevents polymerization of actin monomers
• Aliquot 10 µL 10 mM
• Add 90 uL DMSO (makes 1 mM)
• Dilute this 1:1000 into medium (makes 1uM)

• Dilute the 1 uM to desired working concentration

• Store in the dark, in the freezer

• Adding 490 µL of non-CO2 medium makes 500 µL of 200 µM

• Final dilution should contain no more than 0.1% DMSO

• 250 nM in the dish should give an effect

• 15 - 30 min

Biological Activity for DAPT (Info from Tocris 2634)

Purchased from Fisher: AAJ65864MA

Mfr: Thermo Scientific Chemicals J65864MA

M. Wt: 432.46 Formula: C23H26F2N2O4 Store at +4°C Light sensitive

Solubility: 43.24 mg/mL (100 mM) in DMSO

DAPT is a γ-secretase inhibitor. DAPT reduces Aβ40 and Aβ42 levels in human primary neuronal cultures (IC50 values are 115 and 200 nM for total Aβ and Aβ42 respectively) and in brain extract, cerebrospinal fluid and plasma in vivo. DAPT has no effect on APPα and APPβ levels. DAPT blocks Notch signaling in hybrid human-mouse fetal thymus organ culture (FTOC) and causes ESCs to commit to neuronal differentiation. DAPT can be used in a small molecule cocktail to derive cortical neurons from hPSCs and to maintain hepatocytes in culture. DAPT also promotes the formation of cone photoreceptors in retinal organoids.

β17-Estradiol

Cayman Chemical 10006315

(Purchased 2022 for NAP lab Bio 388)

500 mg

Store powder at -20

Make 0.05M stock solution: 0.068 g in 5 mL DMSO. vortex

Estrogens direct the development of the female genotype in embryogenesis and at puberty. 17β-Estradiol is the major estrogen secreted by the premenopausal ovary. It is synthesized from testosterone primarily in the ovarian granulosa cells and placenta, but small amounts can be produced in the adrenal gland.1,2 Plasma 17β-estradiol levels increase gradually between days 1-7 of the menstrual cycle followed by a sharp increase to a peak value of about 300 pg/ml on day 12, just prior to ovulation

Sigma F1141

Attachment factor

Smo (Smoothened) receptor* agonist

Tocris 4918 10 mg

MW = 459.94

Make a 5 mM stock solution in 4.34 mL DMSO (heat to 50C to dissolve)

*Non-classical G-protein-coupled receptors that belong to the Frizzled family. Smoothened receptors lack the ability to directly interact with their endogenous ligand, Hedgehog (Hh). In the resting state, Smo receptors are bound to patched (Ptc).

Does not recognize the classic cyclopamine (Cat. No. 1623) binding site. Does not promote Smo translocation to the primary cilium; is strongly potentiated by forskolin and cholera toxin. Displays anti-adipogenic effects in vitro, mediated by a non-canonical Hedgehog signaling pathway. Promotes differentiation of multipotent mesenchymal progenitor cells into osteoblasts.

H 89 hydrochloride Tocris 2910

• Protein kinase A inhibitor
• Also inhibits several other kinases (IC50 values are 80, 120, 135, 270, 2600 and 2800 nM for S6K1, MSK1, PKA, ROCKII, PKBα and MAPKAP-K1b).
• Exhibits antinociceptive activity.
• Enhances survival and clonogenicity of dissociated human ESCs through ROCK inhibition.

Store in freezer

1 mg

MW 532.79

1 mg/mL in DMSO

data sheet

Hydroxyurea: (Sigma H8267-1g)

• To make 50 mL YPD with 0.2M HU
  

• vi = (0.2M * 50 mL)/2M = 5 mL

• 100µL 2M stock per mL medium

• Buy 1 g, make 6.6 mL 2M stock
  

• MW = 76, so 1 g = 1/76 mol = 0.0132 mol
  
• vol = (1000 mL/2 mol) * 0.0132 mol = 6.6 mL

See yeast recipes for QBoC https://wahoo.cns.umass.edu/node/554/

TCI H0912

C21H23CIFNO2

MW 375.87

Solubility:¹

• 0.1 M HCl: 3 mg/mL
• DMSO: soluble
• H2O: insoluble
• ethanol: soluble (only in theory - in practice, not so much)

Make 10 mL 10mM stock solution: 0.376g in 10 mL DMSO. Vortex to mix

Haloperidol has been used:

• in ethanol to serves as an inhibitor of Erg2p
• to address the mechanism of haloperidol in ferroptosis using hepatocellular carcinoma cells: Hep G2 and Huh-7 cell lines
• in receptor internalization assay
• as an antipsychotic drug in Dulbecco′s Modified Eagle medium

Biochem/physiol Actions:

Haloperidol is a butyrophenone antipsychotic. It is also classified as a neuroleptic (powerful tranquilizer). Haloperidol acts as a D2, D3, and D4 dopamine receptor antagonist and thus causes Parkinson′s disorder. It also has a negative effect on the central nervous system.

Fisher A16292 (Alfa Aesar) 1 g

1 mg/mL in 100% ethanol.

Insulin-like growth factor-1 receptor inhibitor

Selleck Chemicals BMS-536924

Molecular Formula: C25H26ClN5O3

Formula Weight: 479.96

Solublity: 96 mg/mL in DMSO

Selleck Chemical S7085 (Fisher NC0736836)

IWP-2 is an inhibitor of Wnt processing and secretion with IC50 of 27 nM in a cell-free assay, selective blockage of Porcn-mediated Wnt palmitoylation, does not affect Wnt/β-catenin in general and displays no effect against Wnt-stimulated cellular responses. IWP-2 specifically inhibits CK1δ.

Selective inhibitor of Porcn-mediated Wnt secretion.

IWP-2 is useful in both regenerative medicine and anticancer efforts. IWP-2 inactivates Porcn, a membrane-bound O-acyltransferase (MBOAT), and selectively inhibits palmitoylation of Wnt. IWP-2 blocks Wnt-dependent phosphorylation of Lrp6 receptor and Dvl2, and β-catenin accumulation.

Soluble in DMF at 12.5 mg/mL (26.79 mM) Insoluble in water and DMSO