ES cell medium

DMEM 4500mg/ml glucose, no pyruvate, no L-glutamine

50 ml (10% ) Hyclone FCS (lot tested, special ES stock)
50 ml (10% ) newborn calf serum (lot tested, special ES stock)
6 ml 1X Penn/Strep
6 ml 1x L-glutamine (add fresh every 2 weeks)
6 ml 1x non-essential amino acids
6 ml of 7µ l Sigma β mercaptoethanol in 10ml Hanks Balanced salt solution
120 µl LIF-containing culture supernatent

Thawing ES cells
1. Thaw rapidly at 37˚C.
2. Transfer to 5 ml media.
3. Spin, 1000 rpm, 5 min. RT.
4. Resuspend in 10 ml media, plate on gelatin-coated plates.
(0.1% gelatin in PBS on tissue culture plates, 15-20min.)

Split ES cells
1. Wash plate with Ca/Mg-free PBS 2x.
2. Add 1ml 1x trypsin, 37˚C, 10min.
3. Pipette cells up and down through a 1ml pipette attached to a 200µl pipette tip. Check
to ensure that the cells are single cells.
4. Pellet as above. Plate 2x106/90mm plate. Confluent at 2x107.
5. Passage every 2-3 days. Chnage media as it gets yellow.

Freezes
1. Trypsinize cells as above.
2. Resuspend cells at 5-10x106 /ml in complete medium.
3. Add 100µl DMSO/0.9 ml of cells.
4. Freeze cells at -20˚C, 2hr.
5. Transfer to -70˚C, overnight.
6. Store in liquid nitrogen.

Electroporation
1. Wash cells twice in normal PBS.
2. Resuspend 2x107 cells in 0.8ml high glucose DMEM at RT.
Straight DMEM, no penn/strep, no serum.
3. Add 10µg linearized template mixed with 20µg salmon sperm DNA to the cells.
Transfer into BioRad 0.4mm cuvette (cat#165-2088)
Stand at RT 5min.
4. Electroporate in BioRad Gene Pulser. Single pulse.
250 Volts(0.25constant volts), 500µF, extended capacitance at 100 Ohms, time
constant =10.0
5. Let stand in cuvette for 5 min. after electroporation.
6. Plate each cuvette into 3 90mm plates.
Transfer content into 15 ml tube.

ES protocols Last Modified: p.1

 Rinse cuvette with 2 ml ESgrow medium. Pool.
Plate 1ml into 90 mm plates containing 9 ml ESgrow.

G418 selection for Neomycin-resistant colonies.

1. After 1 day in ESgrow medium, transfer into ESgrowG418 medium.
ESgrowG418= normal ES medium containing 250µg/ml active concentration of
G418.
2. Grow plate until individual well defined colonies are visible.
Mark colonies on the underside with pen.

Picking colonies.
1. Rinse plate with 1ml 1x trypsin-EDTA.
2. Repeat.
3. Scrape colony with sterile P200 pipette tip and 100µl trypsin.
4. Transfer into sterile eppendorf tube with more trypsin.
Pipette up and down to homogenize.
5. Spin at 2000rpm, 2min in a microfuge.
6. Resuspend in 1ml medium. Pipette again.
7. Plate in 1 24 well dish with 2ml medium total. =Passage #2.
Grow until confluent.

First Freeze and make replica plate.

1. Rinse well with 1ml trypsin.
2. Trypsinize in 1ml trypsin. Disperse with P200 pipette tip.
3. Transfer to sterile eppendorf tube.
Spin 2000rpm, 2min.
4. Suck off 900µl supernatent.
Add 1ml ESgrowG418. Resuspend with P200 pipette tip.
5. Plate 100 µl (1/10th of sample) into a 6 well dish = Passage #3.
6. Spin 2000 rpm, 2min.
7. Resuspend in 500 µl ESgrowG418.
Add 50 µl DMSO. Mix rapidly. Place on ice.
-20˚C, 2 hr.
-70˚C, o/n.
Store in liquid nitrogen.

DNA isolation.
1. When the well in 6 well dish is confluent, wash 2x with 1ml Ca/Mg-free PBS
2. Trypsinize with 1 ml trypsin, 37˚C, 5 min.
3. Transfer to Eppendorf.
4. Spin 2000 rpm, 2 min.
5. Discard supernatant. Resuspend in 1 ml PBS. Store at -80˚C.
6. Use ABI DNA extractor to isolate genomic DNA.
7. Resuspend DNA in 300 µl TE.
8. Cut 40µl of DNA with restriction enzyme. Run gel. Southern blot. Probe for transgene.

ES protocols Last Modified: p.2

Expand identified clones.
1. From Southern identify clones that carry the transgene.
2. Thaw only freeze of clone and trasnfer to 5 ml ESgrowG418.
3. Spin 1000 rpm, 5 min.
4. Resuspend in 1 ml ESgrowG418,
plate in 1 well of a 6 well plate. = Passage #4
5. When confluent, trypsinize thoroughly. Resuspend into 10 ml ESgrowG418.
6. Plate 1 ml into each of 3 wells of a 6 well plate, = Passage #5
Plus 1 ml into a 90mm dish.
7. Make freezes from each of the 6 well dish (=3 freezes/clone).

Differentiate ES cells
1. When 90 mm plate is confluent, split 1:10 into second 90mm dish. = Passage #6
2. When second plate is confluent, split 1:10 into 5 90mm dishes. = Passage #7

a) Liquid differentiation.
3. Rinse plates with 2x 5ml Ca/Mg-free PBS.
4. 1 ml 1x trypsin, 37˚C 2min.
Want cells to just start comming off as flakes from the monolayer. Check in
microscope.
5. Add 5ml of ESgrowG418 to each plate to stop trypsin. Pool.
6. Spin 2ooo rpm, 5min.
7. Resuspend into 3 ml of differentiation medium.
DMEM, high glucose. No Pen/Strep. No non-essential amino acids. No β-
mercaptoethanol. 15% Newborn calf serum.
6. Split each confluent plate into 3 plates each.
Add 9ml of differentiation medium to each bacterial plate. = Passage #8
Add 1ml cells.
7. After 4 days, add 5 ml of 20% Newborn calf serum DMEM, high glucose, no
Penn/Strep, no non-essential amino acids, no β-mercaptoethanol.
8. On alternating days, remove 5 ml of medium from plates.
9. Cystic embryoid bodies should start beating around day 10-14 after initiation of
differentiation.

b) Methyl cellulose medium.

3. Rinse 1 confluent 90 mm plate with 2x 8 ml Ca/Mg-free PBS.
4. 1 ml 1x trypsin, 37˚C 10 min.
Pipette up and down with 1 ml pipette attached to a P200 tip. Want a pure single cell
suspension.
5. Add 9ml of ESgrowG418 to stop trypsin action.
6. Transfer to 15 ml conical tube.
7. Count 10µl in haemocytometer. Need to calculate cell number.
8. Spin 2000 rpm, 5min.
9. Resuspend cells to 2.5x105/ml
10. Plate 20µl (5x103 cells) into 1 ml methylcellulose medium in small snap cap tubes..
Vortex breifly.

ES protocols Last Modified: p.3

11. Plate 40 µl (1x104 cells).
12. Let bubbles rise in tube.
Plate content onto 2 cm dish.
Incubate inside 10 cm bacterial dish with a open 2 cm dish of distilled water as
himidifier.

ES protocols Last Modified: p.4