This may be what you were looking for; however, we use trypan blue from (I believe) Sigma
in a phagocytosis assay kit and it seems to work fine. It also list a concentration,
which
a couple of people have asked me.
Cytometry 1994 Dec 1;17(4):294-301
Related Articles, Links
Evaluation of a flow cytometric fluorescence quenching assay of
phagocytosis of sensitized sheep erythrocytes by polymorphonuclear
leukocytes.
Van Amersfoort ES, Van Strijp JA.
Eijkman-Winkler Institute for Medical Microbiology, Utrecht
University, The Netherlands.
A number of reports have been published describing phagocytosis
assays for flow cytometric analysis. In some of these, the
fluorescence quenching technique has been used to discriminate
between adherent and ingested particles. In this report, we
have evaluated the efficacy of a quantitative fluorescence
quenching technique with crystal violet and trypan blue for application
in a phagocytosis assay with polymorphonuclear leukocytes and
sensitized sheep red blood cells. We set the requirements to a
high quenching efficiency of the fluorescence of extracellularly
bound particles and no intracellular quenching. The latter was
determined using polymorphonuclear leukocytes stained with the
fluorescent nuclear dye hydroethidine. We observed that both
trypan blue and crystal violet efficiently quench the fluorescence
of PKH26 (a red fluorescent membrane-associated dye)
erythrocytes but that only crystal violet quenches intracellular
fluorescence. In testing trypan blue and crystal violet from different manufacturers,
there was no real difference between different brands of crystal violet, but only the
trypan blue from Merck turned out to be an efficient quencher, whereas the other
brands of
trypan blue showed low quenching efficiency. Trypan blue at
a concentration of 25-50 micrograms/ml proved to be a good
quencher
of the fluorescent erythrocytes and exerted minimal side
effects: over 90% quenching of the erythrocytes, no intracellular
quenching, moderate increase in autofluorescence of the
polymorphonuclear leukocytes, and no cell loss.
"Alice L. Givan" wrote:
> I cannot, at the moment, find the reference --- but there is a problem with trypan
> blue from certain manufacturers: it doesn't quench external fluorescence even at
> enormously high concentrations. I believe the conclusion was that it might be a
> minor contaminant in the trypan blue that is really doing the quenching. Anyway, I
> certainly used trypan blue at New England clam chowder concentrations and had trouble
> getting quenching. I switched to using Molecular Probes anti-fluorescein antibodies
> --- and got good quencing of external fluorescence (but at a price). Then, after
> the fact, I read the article which explained that I should have looked to a different
> vendor for my trypan blue.
>
> If anyone has the trypan blue reference --- perhaps they could post it to the network.
> Otherwise, I will keep looking until I find it.
>
> Alice
>
> Alice L. Givan
> Director, Englert Cell Analysis Laboratory
> Norris Cotton Cancer Center
> Dartmouth Medical School
> Lebanon, NH 03756 USA
> Tel 603-650-7661
> Fax 603-650-6130
> givan@dartmouth.edu
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