Hi Bernard,
 
I've loaded peripheral blood cells with Indo-1AM for several years using a
variety of methods, for analysis by flow cytometry. I generally want to
look at lymphocytes or neutrophils, and find the best way to exclude
erythrocytes etc from my analysis is by "gating" on loading of indo-1 AM.
They fluoresce much less than mononuclear cells (~10 fold), and I've always
presumed that it's because they can't hydrolyse the ester (a step that is
needed to accumulate indo-1 inside the cells, and also to make it
responsive to [Ca++]).
 
It might be worth while trying to load the erythrocytes with the
non-membrane-permeant form using detergents and/or osmotic shock or even
micro-injection, if the cells can stand it.
 
Ray
 
>On Wed, 29 May 1996, Bernard Himpens wrote:
>
>> We have problems measuring Ca2+ in Red Blood Cells. There is not only a bad
>> loading with fura-2 or Indo-1 but also a very high background fluorescence.
>> Fluo-3 is working better but it is harder to quantify.
>> Has anyone a suggestion to measure Ca2+ in intact cells.
>
 
                              Ray Hicks
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