I've been trying to check the calcium influx in NIH-3T3 cells transfected with a GPCR. The cells are incubated in fura 2AM in 5M concentration in serum free cell culture medium for 30 min at 37°C. I check the viability of the cells before incubating them in fura 2. But after incubation, the amount of live cells (checked by trypan blue) go down and as I proceed with estimation of Calcium ion on flourimeter, the number of live cells almost becomes nil as a result of which I dont get any signal. I incubate the cells in KRH buffer during the experiment. Can anyone suggest me the reason of cell death?
Calcium Ion Estimation