Tricky or stupid question :

I measure in different cell lines Ca-oscillation upon stimuli over a period of 4-6min.

I use FLuo-4-AM dye from Thermo Fisher, btw 3 and 4 microMolar. Incubation in DMEM medium, washing in KRH buffer all according to manufacturer instructions and like former collegues and collaborators do it.

And the measurements themself look nice.

HEK293T cells show without any stimulus, just upon shining the light to excite the dye a pretty synchronized curve (within the first 40sec). Then nothing. With my stimulus the peak has a different shape.

MCK cells do not show such a synchronized peak, however they do show it.

1) why do cells increase their Ca concentration solely upon shining light on them ?

2) what happens to the dye+Ca during the time the peak decreases ?

I am aware of how increase of fluorescence happens : There is dye in excess in the cytosol, as soon as the Ca concentration (due to release from ER or influx from external medium) increases, it binds the dye which increases its fluorescence roughly a 100times.

But what happens when the signal goes down ?

Is the Ca binding reversably, hence Ca that is pumped out of the cell or the ER will unbind from the dye ? Or is it bleaching of the dye, so the subsequent peak (upon my stimulus) would be due to other FLuo molecules binding Ca ions ?

3) I see sometimes that my stimulus leads to a precise compartmentalization of Ca. I see literally tubes (Mitochondria ?!) and then they fade away and the cell is black (dead?)

Thanks a lot for any explanation