5(6) Carboxyfluoresceindiacetate

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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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