Passage 1x onto gelatin or feeders using DMEM-ES media (1-2 days after
thawing mESCs)
Media: Gelatin
Trypsin-EDTA
Wash Media
DMEM-ES
 Gelatinize appropriate # of wells needed to split cells and let sit 20 minutes
 Aspirate media off cells and trypsinize cells with1ml trypsin-EDTA.
 Incubate no longer than 3 minutes at RT.
 Prepare 9ml Wash Media in 15ml tubes
 Pipette cells up/down in their well, using a P-1000, to make a single-cell suspension.
 Transfer cells to prepared 9ml Wash Media.
 Spin at 1400rpm for 5 minutes.
 Aspirate media and resuspend pellet in 1ml DMEM-ES using a P1000 to make a single-cell
suspension.
 Bring volume up to appropriate amount (normally 3ml) with DMEM-ES media
 Aspirate gelatin (or media off feeders).
 Plate cells with ES media at a 1:3 – 1:6 split depending on concentration of the ESCs.
Splitting mESCs onto gelatin to go into differentiation
 Choose best well(s) of cells to carry forward
 Gelatinize appropriate # of wells
 Aspirate media from cells to be split
 Trypsinize cells with 1ml trypsin-EDTA to each well with mESCs
 Incubate no longer than 3 min. at RT
 Prepare 9ml Wash Media in a 15ml tube
 Pipette cells up and down using P1000 to make a single-cell suspension
 Transfer cells to prepared 9ml Wash Media
 Spin at 1000/1400rpm for 5 min (~8C).
 Aspirate media and resuspend pellet in 3ml IMDM-ES using a P1000 to make a single-cell
suspension
 Aspirate gelatin
 Plate cells with IMDM-ES media at a 1:3 – 1:6 split depending on concentration of the
mESCs.