Most of the calibration methods published don't hold up very well because
droplets don't behave at all like cells. Cells have complicated buffering
systems and the Kd of the dyes can change a lot in some cellular
environments. Punching holes in many cell types can be very slow. In my
hands A23187 or ionomycin took 20+ minutes and I fell asleep by then.
Then there's the issue of recoveries which I really like to show to prove
thatI haven't done anything too weird to a cell. The only method that I
ever thought worked was to perfuse a patch pipet with different amounts
of Ca, which was worked out by Don O'malley. unfortunately, you need to
be doing Patch loading.
 
 Yu, et al, J. Neuroscience June94 14(6)3487
 O'Malley, J. Neuroscience Oct 94 14(10)5741
 
 
                                  Barry J Burbach