cell culture plastic: coat with 0.1 -0.2% gelatin for > 10 min at 37C
start mESCs culture
important: use serum+LIF medium for starting the culture !
thaw vial of mESCs quickly, add 1ml of cell suspension to 9 ml of full medium, spin down (7min, 500 RCF), remove supernatant;
optional: repeat wash with 10ml full medium
resuspend in appropriate amount of culture medium and plate
after 1-2 days exchange medium with 2i medium
passaging
passage cells every 2-3 days
wash plate once with PBS (-Mg, -Ca)
add accutase (0.5-1ml per well in 6 well) [important: don't use trypsin to passage cells in 2i!]
incubate 2-3 min at 37C
triturate cells in accutase to break up colonies
add 4ml medium to 1ml cells in accutase
spin down (3-5min, 1000-1500 rpm), remove supernatant; resuspend in full medium
split at ratio 1:5 - 1:16 (typically 1:8)
freeze cells
important: use serum+LIF medium!
resuspend cells in full medium WITH serum
add DMSO to final concentration of 10%
distribute over multiple cryo-vials (1 ml per vial); make sure each vial contains enough cells for a new culture on a 6cm or 10cm dish
freeze cells SLOWLY in Mr. Frosty or in styrofoam container for several days in -80 freezer
transfer cells to longterm storage in liquid nitrogen or freezer with equivalent temperature
culture medium (500 ml)
435 ml Knockout DMEM
50 ml ES certified FBS (10%)
5 ml NEAA
1 ml of 55mM beta-mercaptoethanol
5 ml pen/strep (100x)
5 ml L-Gln (stock: 200mM)
500 units mLIF (50 μl of 1e7 units/ml)
mix ingredients using sterile technique and filter with 200nm millipore vacuum filter (low protein binding!!)
preparation of culture surface, feeder free
cell culture plastic: coat with 0.1 -0.2% gelatin for > 10 min at 37C
glass: coat with 10 μg/ml laminin for 2h at 37C (thaw laminin aliquots slowly in fridge, dilute with sterile PBS)
culture on feeders
plate 0.25e6 - 0.5e6 irradiated MEFs per well of a 6-well plate (for other sizes scale accordingly) in complete medium one day before plating mESCs;MEFs can be used for about 1 week
plate mESCs directly in the conditioned medium;
start mESCs culture
thaw vial of mESCs quickly, add 1ml of cell suspension to 9 ml of full medium, spin down (3-5min, 1000-1500 rpm), remove supernatant;
optional: repeat wash with 10ml full medium
resuspend in appropriate amount of culture medium and plate
passaging
passage cells every 2-3 days
wash plate once with PBS (-Mg, -Ca)
add 0.25% trypsin in PBS/EDTA (0.5-1ml per well in 6 well)
incubate 2-3 min at 37C
triturate cells in trypsin to break up colonies
add full medium to quench trypsin (add 4ml medium to 1ml cells in trypsin )
spin down (3-5min, 1000-1500 rpm), remove supernatant; resuspend in full medium
split at ratio 1:5 - 1:16 (typically 1:8)
freeze cells
resuspend cells in full media WITH serum
add DMSO to final concentration of 10%
distribute over multiple cryo-vials (1 ml per vial); make sure each vial contains enough cells for a new culture on a 6cm or 10cm dish
freeze cells SLOWLY in Mr. Frosty or in styrofoam container for several days in -80 freezer
transfer cells to longterm storage in liquid nitrogen or freezer with equivalent temperature
