Etchevers:Notebook/Genomics of hNCC/2008/09/30

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Freezing cells

2x 35 mm dishes of cephalic and trunk cells - seem to be dying, lots of debris. Trypsinized, perhaps should have left longer (was not at 37 the whole time). Took 27.27 ml of 12% serum-containing Rich medium and added 2.73 ml serum (the Eurobio stuff) to get 30 ml medium. Used 12.5 ml to stop the 0.5 ml trypsin. Then added 0.5 ml DMSO to 4.5 ml of this 20% serum to get 18% serum, 10% DMSO for freezing.

Counts:

  • Cephalic R1066 p23: only 100,000 cells (50 per field, resuspended in 0.2 ml).
  • Trunk R1066 p26: only 144,000 cells (72 per field as above).

Froze in total volume of 1 ml. Placed freezer box (isopropanol?) at -20°C for 1h then transfer to -80°C overnight. Tomorrow must place in liquid nitrogen.

Surviving cells from rabbit injections last Friday seem to be dying too. Highly fluorescent. Some had adhered yesterday and were dying today (see bits of cell membrane left and shrunken cell bodies, plus lots of floating bodies. Perhaps overcharged in CMFDA.

  • Heather 10:29, 30 September 2008 (EDT):


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