Geoff, Very valid points for discussion, too often overlooked. 1. Yes, we tend to check as many fluorochrome combinations possible to ensure we don't either have a problem with missing cells because of low staining, or "finding" additional cells, because of non-specific interactions of fluorochromes. 2. You must, in the end, try all available combinations at some time just to be sure there isn't a problem. 3. Yes, we try ALL possible combinations on cells we know are positive for the markers in question, and cells we know are negative. Together, going the extra mile to test all of these antibody/fluorochrome combinations helps to ensure the data generated are not in some way flawed. It does not guarantee there is not some form of error, but it goes as far as possible. My final thought is that when antibodies are tested, regardless of the tissue system or the fluorochromes present, extreme care needs to be taken with regard to the presence of dead cells. Merely gating out by forward/side scatter can be deceptive, many dead and dying cells have yet to shrink below the size of the viable population and they are very sticky for most antibodies and fluorochromes. Randy >Hi All, > After people's thoughts on choice of fluorocrhome combinations for >various >cell surface receptor staining. > Recently, I asked students to analyse flow data generated from a >matrix of >a titration of both primary and secondary antibodies against a cell surface >receptor which is expressed by a cell line. Then consider the affect >different fluorochromes conjugated to the second antibody has on staining >levels, and our subsequent assumptions about the levels of receptors. > The objects of the exercise were numerous, and the students >questions got >me thinking so I thought I'd stimulate some discussion a few points of my >own thus: >1. Consider this, cells labelled with more than 1 antibody/fluorochrome >combination that bind to recptors on the cell. Do you bother checking each >antibody/fluorochrome combination by swapping fluorochromes attached to >each antibody? >2. How can we sure that the sensitive of our perceived detection system, >that of antibody/fluorochrome, is optimum without trying every combination >of antibody/fluorchorme? >3. If you have more that two antibody/fluorchrome combinations, do you >check each combination without the other ab/fluor. pair(or triplet or more) >present to see if there is "competition" occurring? Steric for example > >That's enough for now, but I'll be interested in people thoughts. > >Thanks >Geoff >====================================================================== >Geoffrey Osborne | ____ __ o Ahh! >Flow Cytometry (FACS LAB) | __ `\ <,_ >John Curtin School of Medical Research, | __ (*)/ (*) >Australian National University, | ==============| >CANBERRA, AUSTRALIA. | |--| >Email: Geoff.Osborne@anu.edu.au | |--|... >Phone: 61 2 6249 3694 | >FAX: 61 2 6249 2595 | >-----Surfing the Web?: Try http://jcsmr.anu.edu.au/facshome.html------ >====================================================================== Randy T. Fischer NIA/NIH 5600 Nathan Shock Dr. Baltimore, MD 21224 (410) 558-8452 (410) 558-8237 (fax) fischerr@vms.grc.nia.nih.gov
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