Hi everybody, IMHO it is important to do all of the controls. I always do the individual labels alone, cells with secondaries only, cells alone (no label) for both flow and confocal cytometry. As far as swapping the secondaries, one needs to use the "strongest signal" for the weakest label,so if there is any doubt, yes, I would do this too. It probably took me a dozen large experiments over a couple of months to get the appropriate labels for a three color confocal experiment. I have also found the hard way that what works for one cell line does not necessarily hold true for another cell line. In other words, there is no such thing as a free lunch. Deb Berglund Microbiology Montana State Unviversity > 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------ > ====================================================================== >
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