Calcium Green-1 AM, intracellular Ca2+ measurement - Do anyone experience with this dye for Ca2+ measuring by spectrofluoro (Nov/12/2007 )

I am now trying to measure an intracellular calcium with Calcium Green1 AM form Invitrogen using Spectrofluorometer.
If someone has experience about this things please help me
I did my measuring based on some published protocol
in formular it used Fmax and Fmin which are fluorescence intensity that used for intracellular calcium calculation
i got a problem with Fmin since they used Ca chelator (EGTA) and MgCl2, can i used the fluorescence intensity from sample with no dye incubated instead because fluorescence intensity of my sample is a bit lower than Fmin which i have optimized for many time but Fmin still got a higher intensity.

here is formula [ca2+] = 190nM (F-Fmin)/(Fmax-F)

I'd be appreciate if someone could help me

i haven't performed this assay but it stands to reason that you would require the dye to determine fmin. this a measure of the intrinsic fluorescence of the dye when not bound to ca. if you leave out the dye then you can't determine this parameter and your calculated [ca] will be incorrect.