I have been working for over a year with this cell type, across two different cell lines and I continue to have this recurring issue. I seed the cells into wells of a 24-well plate and leave in growth media (DMEM, 20% FBS, 1% P/S) for 24 hours. Then I change the media to differentiation media (DMEM, 2% Horse serum, 1% P/S), which I change every 24 hours.
After 24 hours of differentiation some wells will have began differentiating without a problem and others will have cell detachment. After 48 or 72 hours wells that were previously differentiating fine suddenly have the same problem. Other wells I can leave differentiating for weeks and they never suffer said problem. There is no obvious pattern between which wells differentiate without a problem and which ones detach.
At first I thought it was contamination, but I am now convinced it is not, I can cross contaminate all the wells using the same pipette and some will be fine, others not. I have tried seeding at different densities (50000, 100000, 150000) it makes no difference. I have tried not washing the cells with PBS between media changes, no luck. I have seeded the wells using a premade cell mix so that they are evenly disbursed, but the problem persists.
An example of healthy vs detached is linked to this post.
Have you experienced this? Any ideas as to what could be causing it?
Thank you kindly,
Brodie
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