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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