Re: Mast cells and basic orange 21

From: Howard Shapiro (hms@shapirolab.com)
Date: Mon Oct 28 2002 - 23:20:09 EST


Amy Raber wrote-

>I am interested in stimulating a mast cell line and using flow cytometry
>to screen for degranulation.  I came across mention of a dye-basic
>orange 21 that may be appropriate for this assay.  I was hoping someone
>would know where I can purchase this dye?  Is there a protocal somewhere
>for staining?  Or is there a better method to measure degranulation?
>(we are looking into annexin staining for degranulation too)  We only
>have access to 488nm laser.

The "mention" may have been mine.

Basic Orange 21 is available from Sigma-Aldrich (St. Louis;
www.sigmaaldrich.com); it is also known as Astrazon Orange G and the
catalog number is 27,421-6.

In order to differentiate mast cells and/or basophils from other cell types
by the fluorescence of
the dye, it is necessary to use green excitation (515-550 nm), and measure
fluorescence at 575 nm or higher. However, if you have a mast cell line,
you can probably get away with 488 nm excitation; cells' fluorescence
should diminish as they degranulate. It is best to stain the cells with
fairly high concentrations of dye (10 micromolar or above); it occurs to me
that you might get better differentiation of cells that have and have not
degranulated by using a ratio of long to short wavelength fluorescence
(e.g., 575 nm to 530 nm) than by measuring emission at a single wavelength.

There are a number of papers on measuring basophil degranulation by flow
cytometry, e.g.:

Brown V, Ennis M: Flow-cytometric analysis of basophil activation:
inhibition by histamine at
conventional and homeopathic concentrations.Inflamm Res  2001 Apr;50 Suppl
2:S47-8

Belon P, Cumps J, Ennis M, Mannaioni PF, Sainte-Laudy J, Roberfroid M, Wiegant
FA: Inhibition of human basophil degranulation by successive histamine
dilutions:
results of a European multi-centre trial.Inflamm Res  1999 Apr;48 Suppl
1:S17-8

These are a bit controversial because of their association with what many
people regard as not completely unbiased attempts to demonstrate effects of
vanishingly small concentrations of agonists and antagonists on
degranulation; more to the point, they may not apply to mast cells, but you
could take a look, and/or look at the references these authors cite for
methodology.

In my limited experience, it may take several hours for cells exposed to
degranulating stimuli to exhibit a notable change in Basic Orange 21
fluorescence. This came as a bit of a surprise, because histamine release
is supposedly demonstrable after a much shorter time interval. I am still
interested in the phenomenon; I haven't had much opportunity to examine it
further because I have been way too busy with other things.

-Howard



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