Anyone have any experience with in vitro differentiation of neural stem cells at various plating densities? I have found that plating at densities within the usual recommended range (50-100,000 cells/cm2) does not give an adequate amount of RNA from TRIzol extraction.
However, from what I understand, too high of a density may inhibit differentiation. I am planning on trying 100-500,000 cells/cm2. What upper limit have you found, if any, where neural stem cells are unable to differentiate as effectively?
We have used 300,000 cells/cm2 and they started differentiation (we cultivated them just for few days), though I do not have comparison of effectivity with lower densities.
We use 120,000 cells/cm. We use 6-well plates and make RNA from 3 individual wells. This works well with Trizol.
Quantity, quality and survival of iPSC-derived neurons decreases with low densities.
Seeding density is important..as with the comment above we haven't done a study of different densities but I use a lower plating density for human NPCs than I do for mouse or rat. For human cells I generally use similar to above, about 100,000cells/cm2. For mouse and rat I would use about 150,000-200,000/cm2. That seems to work for us. Just a tip with the trizol (if your not doing it this way already!)...don't scrape/trypsinize the cells then add to trizol (unless there is a specific reason you have to do this)..we add the trizol directly to the cells after removing the media..works better, the cells lyse straight away.
As Pavel Ostasov said, I commonly seed 300,000 cells/cm^2 and they grow well, but if you want an extraction, You can wait until the first 4-5 days. And what was your medium? Because you can add different concentration of FBS and this is going to modified your final concentration of your cells for the extraction. All in a flask of 75cm^2! good luck!
- Badges
- Science topic
- Similar topics
- Indexes