Etchevers:Notebook/Genomics of hNCC/2009/03/06

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Freezing cells

Trypsinized with 10 mL freshly thawed trypsin the two 150 cm2 flasks - one in regular Rich medium (R), one in Rich medium prepared with Stemline Neural instead of DMEM/F12. The cells look the same overall.

Counts: S - 202 in 3 of 5 rows = 202 x 5 / 3 / 2 x 104 cells = 1,683,333 cells in 0.5 mL.

  • Took 74 μL for a 25 cm2 flask (collagen on this morning, rinsed abundantly in PBS)
  • Needed 300 μL (1 million) to freeze

R - 246 x 5 / 3 / 2 x 104 cells = 2,050,000 cells.

  • Took 55 μL for a 25 cm2 flask
  • Needed 250 μL (1 million) to freeze.

Diluted each freezing volume with one volume of 20% DMSO in Rich medium. Placed in isopropanol freezer helper pre-cooled to 4°C, then at -20°C at 5PM.

The rest of the cells were re-pelleted, and resuspended in RLT + β-ME lysis buffer for RNA extraction, and placed in the freezer as well. If you can expect about 10 μg per 106 cells, here I can hope for 8 μg from the Rich cells and for 4.2 μg from the Stemline-Rich cells. These are in the culture room freezer for now with the serum in the bottom of the drawer.

Trypsinized the two neural differentiation formulations, diluted by 1/2. The counts were:

  • B27 1x + vitamin A (retinyl ester) but no other adjuvants aside from glut/p/s : 570,000 cells in two 25 cm2 flasks and some of the unattached-in-1-hour supernatant transferred to two collagen-coated coverslip wells of 24-well dish.
  • B27 1x + 0.2 ng/mL FGF2, 0.1 ng/ml EGF: 550,000 cells also in two 25 cm2 flasks and some of the unattached-in-1-hour supernatant transferred to two collagen-coated coverslip wells of 24-well dish.
  • Heather 12:36, 9 March 2009 (EDT):