Ah the old minus [Ca++] trick. The assumption when trying to measure Fmin
is that by treating the cells with ionophore and using a chelator the all
of the calcium gets out of the cell and therefor the Fmin represents the
fluorescence intensity when [Ca++]=0. In reality, the chelators are too
big to enter the cell through the ionophore holes in the membrane so the
calcium in the cells does not go to zero. If the resting [Ca++] is high,
then you get a great reduction in "Fmin" with the ionophore/chelator and
the numbers look OK. In reality, many cells have a very low resting
[Ca++] and measuring the Fmin this way does not work. Correctly
estimating Fmin is very hard to do and usually involves waiting a long
time after adding ionophore and chelator. You may also need to add
something like Thapsigarigin (sp?) to release internal Ca++ stores.
Contact Ole Thastrup at Novo Nordisk in Coppenhagen.
 
________________________________________________________________________________
 
 
Paul Goodwin
Image Analysis Lab
FHCRC, Seattle, WA
 
On Fri, 24 May 1996, Malgosia Szewczenko-Pawlikowski 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!
>