Cells are unhealthy - (Mar/13/2014 )
I am culturing the fibroblast cells. My cells become unhealthy every a few generations. But my colleagues are OK. The only differences are the incubation and the FBS and Pen strep concentration. I put exactly 10% FBS and 1% Pen Strep. They just put 50 mL and 5mL respectively to the medium. For me, I put 56.1mL and 5.61mL. Why do I ask here because I am wondering the affection of a little higher FBS and Pen Strep is so much! I paid attention all of the tricky stuffs for cell culture. Somebody can give me some suggestions or something else?
Of course, I will try to low down the FBS and Pen Strep concentration as my colleague is doing to see what happens.
Well the logical thing to do would be to try using their conditions...
However, depending on the line (immortalized/transformed or not) cells do reach a point known as the Hayflick limit, where they reach will stop replicating and enter senescence, never to divide again. This could be what you are experiencing.
Since your coalleague's cells cultured at the same time are ok, I don't think the problem is that your cells are reaching senescence. I also don't think those extra microliters of FBS and P/S would make a big difference. I think the devil is in those "tricky stuffs". Is your colleague more experienced with cells? Can you ask him/her to watch over your shoulder when you are working with the cells and tell you exactly what (s)he would do different?