1 million cells loaded with indo-1AM were put to cuvette in Hitachi F-2000
fluorescence spectrophotometer with an excitation wavelength 355nm and
emission was detected at WL1=410nm (for Ca- bound indo-1) and at WL2=485nm
(for Ca-free indo-1).
To obtain Fmax cells were treated with 10 microM ionomycin and 4mM calcium.
To obtain Fmin cells were treated with 2mM manganese (Mn) and Fmin was
calculated from equation:
Fmin=[(Fmax-FMn)/12]+FMn
Having values of F1, F2, Fmax1, Fmax2, Fmin1 and Fmin2 calcium level was
calculated from Grynkiewicz's equation:
[Ca]i=K{[(F1/F2)-(Fmin1/Fmin2)]/[Fmax1/Fmax2)-(F1/F2)]}(Fmin2/Fmax2)
In all results the ratio Fmin1/Fmin2 was bigger than F1/F2, so final calcium
concetration always has minus values.
What I would like to know is if the above eguation for Fmin has an error
that results in these negative values.
PLEASE HELP!