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------ ======================================================================
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