Answer:

I'm currently using RPE1 cells, and I am having big PROBLEMS to make them grow. The people around me try to help me TROUBLESHOOT, but we have not been able to determine why these easy cells will not grow for me.

We are all using the same cell line, the same serum, the same media, flasks, incubators etc. I've looked over the others' shoulders and they over mine. The one thing I have not been able to compare with them is the seeding density, since none of them counts their cells. They just pass them at 1/10 for 48 hrs or 1/15 over the weekend.

What I (think that I) need to know is:

- when to pass the cells, i.e. what 80% confluency looks like (if 80% is good to pass). A picture would be wonderful! Or a comment on this image (good to pass, under confluent or over confluent

Explanation:

Ask a colleague to split one of your cultures using your supplies and reagents. Ask a colleague for one of his/her cultures and split it using your colleague's supplies and reagents. Keep the TWO sets of cultures in the same incubator - preferably on the same shelf.

If the cultures that were handled by your colleague grow well, but the cells handled by you do not grow, the problem is YOU. One former member of my lab had this problem. It turned out to be caused by rough handling of single cell suspensions (pushing and pulling them through narrow gauge-needles with excessive force.) By the time the cells were aliquoted into new culture plates, most of them were dead. Therefore, if the first round of testing indicates that YOU are the problem, determine how many of the cells are still excluding trypan blue when you get ready to plate and how many are ADHERING to the surface of the culture flask after a reasonable period of time