Etchevers:Notebook/Genomics of hNCC/2009/01/22
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Thawing out early passage CCNCh
Sophie mailed me lots of things, among which a new aliquot of hNCC cephaliques. Made up new medium with new F12/DMEM and no BjGb - DMEM instead. Counted 90 live cells in central of 9 squares of Neubauer, as well as 8 dead (trypan-blue) absorbing cells. This was after resuspending in 500 ul and diluting 1x with the trypan blue (1/10 in PBS prepared by Magali). Meaning 98 cells total, and my rule of thumb is to multiply by 104 - this is 980000 cells, very close to the million I should have.
22 January 2009:
thawed cells which were frozen at P4 (considered plated as P5, perhaps wrongly). Rich medium now no longer has BjGb but only DMEM and F12 in addition to 12% serum and all the other adjuvants. Need to type up recipe for dissemination.
Approximately 1 million cells to start – counted 90 live + 8 dead cells in Neubauer hemocytometer. These spread to confluence in 35 mm dish by the next day (theoretically a concentration of 450,000 cells per 9.6 cm2 or 47,000 cells per cm2. 100mm2 = 1 cm2.