did you add Cobalt to your working solutions?

We only have experience of Calcein AM staining of low density, primary neuronal cultures from embryonic and neonatal mice. We use Calcein AM to allow us to take images of neuronal processes for analysis of their length and branching. Over the short term (12-24 hours) Calcein AM does not seem to kill neurons. Some minimal neuronal death is seen by 48 hrs of culture with Calcein AM and this is increased by 72 hrs culture. It should be pointed out that at low density, embryonic and neonatal neurons are quite vulnerable to any trauma and die easily, so this may suggest that if these cells can survive for 24-48 hrs with Calcein AM without significant cell death, more robust cells/cell lines will be less affected and may be impervious to any toxic effects of Calcein AM.

I did not add cobalt to the working solution. I tried staining the cells with Calcein AM then incubated for several hours. Some cells were dead, some were still alive. Thanks for all the answers.

So It can be an osmolarity artefeact of your incubation solution. try to  add an aliquot of cacein AM directly to the culture medium.....

May consider using Permai fluorescence dye.