Do you have a reason to use flow rather than histamine release for these assays? Since these are cell lines I assume you will not be using the multiparameter capability (useful for gating on the <1% of cells that are mast cells or basophils). By using a less-than-standard and not well accepted assay you are asking for a reviewer to roast you alive. One major problem with flow assays of degranulation is that they typically do not give you data expressed as "% total degranulation". Well accepted degranulation assays, such as histamine or beta-hex release, are always expressed as a fraction of the release obtained after mechanically disrupting the cells with detergent or such. Howard, I assume basic orange 21 stoichiometrically binds to acidic granule proteins in the mast cell and since there are fewer granules after degranulation, that reads out as lower fluorescence. Correct? Another major problem with such flow assays is that if you are measuring degranulation by drop in fluorescence of intracellular granule protein (such as tryptase or by basic orange 21 binding) your experimental "window" is likely to be quite small. For example, if you get 20% histamine release (say 5% release with media, 25% with activation), that would read out as a 20% drop in mean fluorescence intensity, say from 1000 to 800 fluorescence units. Not exactly a robust response. CD63 translocation (basotest) reads out more like a conventions degranulation assay. As one who has been on the spit for this and doesn't wish to see you suffer, my advice is to use the tried and true. Unless the major focus of your work is the development of new flow assays. If the only tool you have is a hammer (or a flow cytometer), every problem is a nail (or a cell waiting to be stained with mAb). Calman > ---------- > From: Amy Raber > Sent: Wednesday, November 6, 2002 10:59 > To: Cytometry Mailing List > Subject: degranulation-another question! > > > Hello everyone, > > First let me thank everyone for all the responses to my question > regarding mast cell degranulation detection by FACS. I am in the > process of trying several of your suggestions and tracking down the > mentioned references. > > Of course I have another question... The researcher working with these > cells wanted to know if any of you had a standard degranulation protocal > > that has worked well. And if so how much degranulation should we expect > > to see? 20% 100%?? We are working with a mouse cell line MCL 9 and a > rat line RBL-2H3. > > Thanks again for all the help! > > Amy > > Amy Raber > Flow Manager > Athersys Inc. > Cleveland, OH 44111 > 216-431-9900 ext 280 > araber@athersys.com > >
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