Etchevers:Notebook/Genomics of hNCC/2009/01/27

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Passages and counting

27 January 2009

Cells look happy on 35 mm dishes but bigger, more fibroblast-like and often with a small thin prolongation with a lamellipod at the end.

Same in the 24 well dishes, nearly confluent in middle and not on edges. Part optic and part liquid circulation effect, leading to highly different cell concentrations.

Left 1x column with Rich and 1x column with Neural differentiation media, at the original seeding densities from last Friday 22/1/09.

Trypsinized 3x wells for uptake, count and reseed into a 4th 35 mm dish. If these are 9.6 cm^2 and I have 150 cm^2 flasks, will need to grow to confluence to seed into a flask, and 5 x 35 mm dishes would be better for this purpose.

Counts: 11 μL of 50 μL used to redilute gave this, leaving (40)μL in each of four cell concentrations.

  • Top row: 173 (x 10^4 / 20 /3 or 167) = 28,883 cells per well
  • 2nd row: 306 x 167 = 51,102 cells per well. Must have mistaken which well was which? Or density growth inhibited for top row?
  • 3rd row: 4 x 167 = 668 cells per well.
  • 4th row: 5 x 167 = 835 cells per well.

Globally, this is also the impression I had, that in 4 days the low density-seeded wells hardly divided with few duplets; the highest density could not grow anywhere, so 2x a week, at the middle density, there is a population doubling. This density is approximately at 10-15K cells per cm^2; to have enough to seed into a 150 cm^2 flask, would need 1,500,000 to 2,250,000 cells.

What was left was re-plated into a 35 mm dish, passage 6 for real as I miscounted from the beginning. Corresponds to (28,883+51,102+668+835)x3 x(0.8, the residue after counting), or 195,570 cells – and eyeballing this they seem like quite a few. The other 35 mm dishes are at passage 7 since yesterday.

So, after thawing and a few days, now have approximately the same number of cells, but they are at a good plating density in 4 x 35 mm dishes. Could come in and pass them on Saturday.

  • Heather 05:58, 30 January 2009 (EST):