Hello!

I am currently working on a cell culture line from embryonic rat fibroblasts, a simple cell culture that doesn't need so much. The lab protocol says to renew media in them two times a week, but also to split them two times a week. I do both at the same time since they grow so fast so ergo every time they are worked on is twice a week.

We have too many flasks with fibroblasts in the incubator and would like to freeze the cells. I attempted to freeze them under the assumption all cells are viable. However, when using Trypan Blue it proved otherwise. When I used a hemocytometer to analyze the cells (pipette trypan blue with cells, 1:1 ratio, under cover slip), the north island is high in viability, but the South Island is all blue cells. 

Is there something wrong when I clean the cells or when I used the hemocytometer? I was told that there may be a problem cleaning the cells but maybe it is in the viability procedure with Trypan Blue.

Any and all help is appreciated. Thanks!