You Wrote:
>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!
 
If I understand your equations properly You determined Fmin By dividing Fmax
by 12?. While this might give you an estimate of Fmin it will not be an
accurate value of the Fluorescence at 0 Ca. You should normally add excess
EGTA to the cuvette after determining Fmax to set the calcium level to zero,
That will then be Fmin. The manganese quenches Indo Fluorescence and give
you a correction for cell autofluorescence or any extracellular indicator.
Even if you add EGTA there are no guarantees that you will set [Ca] to zero
, the same problem is true for setting Fmax but you should usually be able
to get [Ca]i above the saturation point for indo (~ 10uM).
The value of the Kd may also be different for your cells so it should be
determined for your conditions in your cells.
There are other factors that can cause negative [Ca] values e.g. binding of
the Indo to intracellular proteins or membranes, or "viscosity effects".
However, these are not normaly problems when doing an "in vivo" calibration
in a spectrofluorimeter. Also bear in mind that even the best calibration
has some errors and will only give estimates of the intracellular [Ca]
Hope that helps?
John Cork
Calcium Imaging Facility
Department of Anatomy, LSU Medical Center
New Orleans, Louisiana 70112
Tel (504) 568 7059       Fax (504) 568 4393
e-mail  ---  [log in to unmask]