Hi,
I am trying to subclone iPSCs by plating 200-300 cells in 6 wells previously coated with geltrex. When I plate them I use 10uM of rock inhibitor and in theory I j
keep it until a nice colony is formed. I tried both E8 and stem flex media. however the day after I plate them, I got single cells but then after 2-3days they die or they remain as single cells without proliferating. I Have tried to change their media every other day as well as every day (I thought maybe the rock inhibitor at 37 degree got degraded). Any suggestion?
The cell seeding density of 200-300 cells per 6 well is probably to low. iPSC like adjacent cells for better survival. You could try 48 or 24 well plates and/or more cells. The thawing AND freezing protocol should be optimized. iPSC should be frozen approximately 2-4 days after passaging. Freezing them significantly later after the last passaging, strongly decreases cell survival after thawing. Please find further information attached.
- Badges
- Science topic
- Similar topics
- Engineering
- Materials Engineering
- Powders