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> Hello all,
>
> A colleague who is not on the mailing list for this group wants to
> measure pH in endosomes and lysosomes using a FACScan flow
> cytometer. She's been looking for protocols without much success.
> Can anyone help? Information can be sent either to the list (for
> anyone else interested in the procedure) and/or to her directly to
> her at natale@ivi.ch . Her name is Valerie Natale. Cheers.
>
> Annette Arriens
>
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Annette, Valarie, and flow list:
We attempted to ask this question using FITC-labelled antigens taken
up by normal mouse B cells in vitro. This was published in Mol. Immunol.
28:779 (1991). Basically we used the fact that chloroquine can neutralize
the low pH of endosomes, and developed a fixation technique with ethanol
the maintained all the internalized FITC-label. We found that in
solution, the fluorescence of our FITC-antigen was altered by a factor of
six, when pH was adjusted from pH 5.5 to pH 7.2. After internalization
(and stripping of surface bound material) chloroquine could only effect a
3 fold shift in fluorescence intensity. However, the same cells fixed in
ethanol and exposed to pH 5.5 and pH 7.2 had again the 6 fold shift.
This indicated to us that not all of the internalized material made it to
low pH compartments.
The work was done on a modified FACS II instument, but I'm sure the
FACScan is sensitive enough to do the same. This may be a useful technique
for your work, provided that you have a selective FITC marker that is
internalized to the compartment of interest. I'm not sure this
technique is capable of clear quantitative work, and I never pursued it
as such.
Denis Snider PhD
Associate Professor
McMaster University
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On Mon, 20 Nov 1995, Annette Arriens wrote:
> Hello all,
>
> A colleague who is not on the mailing list for this group wants to
> measure pH in endosomes and lysosomes using a FACScan flow
> cytometer. She's been looking for protocols without much success.
> Can anyone help? Information can be sent either to the list (for
> anyone else interested in the procedure) and/or to her directly to
> her at natale@ivi.ch . Her name is Valerie Natale. Cheers.
>
> Annette Arriens
>
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