Mouse ES Cell Handling

Recipes for Media

ES Cell Medium

 To a 500ml bottle of DMEM (Specialty Media) add:

 3 ul 2-mercaptoethanol
 5 mls 100x gentamycin sulphate
 5 mls 100x glutamine
 50 ml fetal bovine serum

STO Cell Medium

 To a 500 ml bottle of DMEM (Specialty Media) add:

 5 mls 100x gentamycin sulphate
 5 mls 100x glutamine
 50 ml bovine calf serum

Freezing Cells

Harvest cells as usual.  Pellet (1,000 r.p.m; 5 mins).  Aspirate supernatant and flick tube to loosen cell pellet.  Add (cold 4°C) freezing media (10% V/V DMSO/serum aliquot and place in Styrofoam box at –70°C.  Transfer to liquid nitrogen the next day.

Passaging Cells

Prepare STO feeder cells in advance:
Mitomycin treatment (from a 10 cm dish you get approximately 6-9 x 106 cells):
1. To each confluent dish, add fresh medium (5ml) and 50µl of mitoC 100x stock sol.
2. Incubate at 37°C for 3 hrs.
3. Coat dishes for >15 min with 0.1% gelatin (made up in ultrapure H2O from a 2% stock)

Plating feeder layer:
1. Aspirate mitoC, wash cell layers 3x with 10 ml PBS
2. Trypsinize mitoC-treated STOs, stop trypsin by adding 2 ml medium/dish, fill volume with PBS to 10 ml
3. Count cells, centrifuge suspension, resupend pellet in medium (ES medium if feeders are used immediately); adjust cell density to 3 x 105/ml
4. Aspirate gelatin solution from coated dishes.
5. Plate 10mls/10cm dish;  3.5 mls/6cm dish;  0.5ml/24 well plate;  100µl/96 well plate.
6. Incubate cells for >3h

Plating ES cells:
1. Trypsinize ES cells, passage confluent dish at 5-fold dilution (1:4) on feeder cells
2. Feed cells with fresh medium 1-2x per day
Next passage will be required after 2-3 days

Reagents

hygromycin B:  Roche # 1084355501, liquid 50 mg/ml stock (1.1 ml/500ml bottle of media)

G418:  Gibco/BRL Geneticin #11811. 100x stock (needs to be adjusted according to effective concentration)

Gentamycinsulfate: Sigma, G-1264 (5g)
(rantine antibiotic) to make 100x stock, dissolve in finally 3.24 ml H2O
 (i.e. effective conc. 10 mg/ml)

DMEM, H2O, PBS w/o Ca, Mg:
 Specialty Media Inc.
 245 Bryn Mawr Ave.
 Lavallette, NJ 08735

Mitomycin C: Sigma M-0503, 10 x 2 mg
 100x stock sol.: dissolve 2 mg in 2 ml H2O using a syringe

Gelatin, 2% stock: Gelatin: Sigma (G-1890) Swine Skin, tissue culture tested, open fresh 1 liter bottle of ultrapure H2O, dissolve 20 mg gelatine powder in 200 ml by autoclaving.  While still hot (i.e. not solid), poor back into water bottle
 to give 1 liter of 2% sol.; store at 4oC
 
Gelatin, 0.1%: melt 2% stock in 65oC bath, dilute with ultrapure H2O to 0.1%