9/15/18

General Guidelines for growing J1 ES cells

IMPORTANT: To ensure the maximum probability that cell lines will contribute to the germ line, it is absolutely necessary to follow as closely as possible, the media, cell density, and other conditions recommended here. Keep the cells happy, and they will perform.

-Only use very freshly made DMEM for media

-Always use gelatanized dishes and flasks

-Always plate ES cells on gamma-irradiated feeders (EF cells) that are plated at least 4 hours before ES cells (unless ES cells are to be harvested for DNA preps). Always check to make sure feeders look well spread and healthy before plating ES cells on them.

-Plan to split every other day.

Important numbers to help calculate ES cell plating densities

-cells should be plated at approximately 60,000 to 64,000 cells/cm2

-confluent ES cells are approximately 2.5 x 105 cells/ cm2

-approximate areas, confluence cell counts, and plating ranges of commonly used dishes:

Approx. cell # atPlating densities

DishArea (cm2)ConfluenceIdealRange

T2525 6.25 x1061.6 x106

24 well1.8 4.5 x1051.2 x105

6 well9.6 2.4 x1066.1 x105

10 cm78.5 2 to 4x1075 x1064 to 7 x106

Important: Double check cell numbers you get on splits with these to make sure you are within these recommended guidelines.

Trypsinizing ES cells from 24 well plates

REMEMBER: THE GOAL IS TO GET SINGLE CELLS. SO FOLLOW THE TRYPSINIZATION PROCESS BY LOOKING IN THE MICROSCOPE TO ENSURE THAT THE MAJORITY OF CELLS ARE SINGLE CELLS.

Remove media, wash 1x with .05% tryspin in DMEM, add .25% trypsin (100 ul if 1 well of a 24w, more if larger plate), incubate 4 minutes 370C in CO2.

Add more .25% trypsin (100ul for a 24w) Pipet up and down to get cells off of bottom of plate. Pipet up and down @20 times with a P1000 and a stuffed tip (set at 150 ul for a 24w). (This trituration with a P1000 is much more affective for dispersing cells into single cells than trituration with a big 1 or 5 ml pipet)

Put back in incubator for @3 minutes more. Pipet up and down again. Add media to stop trypsin and collect cells in centrifuge tube.

Spin down cells.

For freezing cells, freeze at @ 4 X 106 cells/ml in 10% DMSO

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