Etchevers:Notebook/Genomics of hNCC/2009/01/26

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Cell counts with different seeding densities

26 January 2009:

Replaced last column with 100% Stemline medium. Replaced medium in columns 2-5 with 100% fresh Rich medium.

Trypsin for 3 minutes with 0.2 ml warm trypsin after 2x 0.5 ml rinse with warm PBS. All cells seem well detached, but clumpy, and stopped solution with 50 l of serum.

Stephane counted a direct sample of the first two wells of the first column. The top well was already confluent, the second well probably 80% confluent. The bottom two are quite sparse, essentially clonal or in two’s.

Cells look best at the “25K” (16830) cells/well = 200 mm^2 – or 2 cm^2 (as opposed to 9.6 for the 35 mm dish). This is a density of about 8500 cells/cm^2, whereas they seem confluent at 15,000 cells/cm^2 and of course, even more so at 45,000 cells per cm^2.

First well: volume of 0.25 ml, count of 8 cells per ninth square = 20,000 cells per well in first well. About ½ in second row, 3 cells, 7,500 cells per well. But I decided to remove all cells, resuspend in 20 l (1/50 mL) and count as such. When I did that, I saw that some cells remained on bottom of all wells.

  • Row 1: 147 cells / 2 squares counted x (10^4 cells/mL x 1/50 mL or 200 cells) = 14700 cells
  • Row 2: 233/8 x 200 = 5825 cells
  • Row 3: 12/9 x 200 = 267 cells (problem!)
  • Row 4: 22/9 x 200 = 489 cells.

Passed the newly confluent P6 cells in 35 mm dish into 3x 35 mm dishes. On counting, 166 x 2 squares counted x (10^4 cells/mL x ½ mL) = 415,000 cells. This means seeded 138,000 cells per dish (9.6 cm^2) or 14375 cells/cm^2. This is similar to the “good-looking” density in the second row of the 24 well plate. However, I don’t know why the counts are so off in said plate; they are far too low.

What is the minimum number of cells can seed to pass only 1x per week eg. without losing the phenotype?

  • Heather 05:55, 30 January 2009 (EST):


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