I have been culturing neurospheres generated from neonatal mice. To summarize what I've done with them...
1. Started a primary culture
2. Treated them with lentiviral shRNA
3. FACS sorted them
4. Put the sorted cells in culture (about 8,000 cells per mL. I have them in 24 well plate)
My question is : Why aren't they growing as fast as they used to?? It's been over a week and I need to expand them so that I'll have enough cells for the subsequent assay... How much does the seeding density affect the growth of these cells?? They don't appear to be dead and they are not differentiating either, and I'm pretty sure they are still alive, but they are just not growing as fast as they used to... Would passaging them help them start growing faster (although I don't think i have enough cells to passage... afraid I might lose more than I will be left with... ) ??
Any advice would be appreciated!
Thanks in advance!