Hi! I would like to perform a doubling time assay in several newly derived human embryonic stem cell lines. Since hESC must be passaged as cell clumps, I cannot know the exact number of cells I am plating. For the assay I need to seed cells in more than one well and count them at different time points. Since the number of plated cells will not be exactly the same in both wells, I am afraid this could affect my results. If you could give me some advice I would be very grateful, thank you!
Hello, Ot!
Please You look at following article:
Article Comprehensive characterization of four different populations...
Proliferative potential of MSCs
The 4 populations of MSCs beginning from passage 1 were trypsinized from the culture discs before plating into the 6-well plates at a density of 105/well. Each population of MSCs was seeded in triplicate. Every 4 days, the cells were harvested with trypsin/EDTA and counted using a hemacytometer for up to 8 passages. The mean value of the cell number counts was calculated from 7 tissue samples from each cell population and the mean population doubling time was obtained for each passage according to the following formula: population doubling time = T x lg2/(lgNt - lgN0), where T is the culture time, N0 is the initial cell number and Nt is the harvested cell number.
This sounds weird. hESCs do not HAVE to be passaged in clumps. As far a s I know, you can dissociate them with accutase of even Trypsin, then count, and seed. Seeding should be very dense, but that's all.
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