Dear Scientists,

I have a question for you. I am using the INS-1 cell line. I am trying to find a kill curve in this cell line with a zeocin at 0, 50, 100, 250, 500, 750, and 1000 μg/ml . I change the media I prepare with this antibiotic every three days and perform MTT. I am seeding 96 well plates. The procedure states that the cells should be seeded at 25% confluency. I seeded eight thousand cells per well. When I look at my MTT results, I see that my cells are not dead.

My question for you is:

1) When I counted the cells on the Thoma slide, I saw about 35, but they are quite dense when seeded. How can I overcome this problem?

2) What is seeding cells at 25% confluency? How can I understand this concretely? Because the cells I count and the cells I seed are not the same.

3) How should I change the concentrations of the antibiotic I use for the MTT study?