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With this reagent, the diacetate is cleaved intracellularly, at which point
the fluorescein becomes flourscent, and is retained inside only live
cells......for awhile. Eventually the cells leak, and the fluorescent
compound can get into dead cells as well. One needs to perform the
analysis ASAP after staining, I used to wait only five minutes or so.
Hope this helps you, don't know why you are having clumping problems.
Deb
On Fri, 13 Sep 1996, H.-Joachim Schuberth wrote:
>
> Dear flowers,
>
> recently a thread dealt with fluorochromes for cell tracking experiments.
> We tried 5(6) CFDA obtained by Sigma, dissolved it in DMSO, diluted it
> further
> in PBS and store aliquots at -20oC
>
> Problems:
>
> (1) bovine and canine Cells (lymphocytes after ficoll isopaque separation
> or leukocytes after hypotonic
> blood lysis) tend to aggregate very seriously when labelled in PBS w/o
> Ca2+, Mg2+
> WHY ?
>
> (2) not only viable, also (sometimes) dead cells are labelled !
> WHY?
>
> (3) cells loose their fluorescence very quickly (2-4 days after labelling)
> WHY
>
> Obviously, I have made some seroius mistakes
> could someone please enlighten me ?
>
> thanks in advance
>
> Hans-Joachim Schuberth
> Immunology Unit
> Veterinary School
> Bischofsholer Damm 15
> D-30173 Hannover
> Germany
>
>
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