Lorrie, It depends on what you mean by success or failure! Certainly there are reports in the literature on the use of CFSE and other dyes to track proliferation of cells. We've been using the same system here to look at proliferation of cells from macaques. This works beautifully for basic research and has been quite helpful in looking at what cells are proliferating as opposed to the total proliferation as assessed by thymidine. BUT, if you're talking about clinical application of such work, then there is one major problem with these flow-based assays. Data analysis. Thymidine is "easy" - you take your radioactivity values and compare wells. But data analysis can be huge with CFSE. Suppose, for example, you want to track proliferation of CD4 and CD8 T cells. Easy enough to gate based on CD4 vs CD8 and then do histograms for CFSE expression in each population. Then you have to determine where your peaks are (at least 8-9 possible peaks for CFSE) and the percentage of cells in each peak. You can then print that out and type it into Excel and use a template for quickly determining the frequency of responding cells or the division index, depending on which reference you've read on these assays. I can't as yet decide which measure is more 'accurate' for what we want to know. As an example - we've been doing this on 96 well plates using the Multiwell autosampler on a FACSCalibur. We have typically done duplicate wells with 4-5 dilutions of antigen plus two control (media alone) wells. So for three antigens we need 30-36 wells. So we can get two, or at most three, animals per plate. Acquiring the data (30k events) takes half a day for 60-70 wells, the analysis of which can then take up easily a full day (from printing out the plots on WinMDI to entering the data into an Excel template). I'm getting better at the analysis end of things and I'm hoping to get software capable of batch analysis so I can automate the analysis end of it as well. Either way, though, it will be time-consuming. My opinion (for what it's worth!) is that these assays are great for basic research but until the time required to do the analysis is shortened I don't see much potential for clinical application, at least not for large numbers of samples. -Doug Douglas S. Reed, Ph.D. Microbiologist Respiratory & Mucosal Immunity Department of Aerobiology & Product Evaluation Division of Toxinology & Aerobiology U.S. Army Medical Research Institute of Infectious Diseases 1425 Porter Street, Fort Detrick Frederick, MD 21702-5011 301-619-6728 301-619-6911 fax doug.reed@det.amedd.army.mil -----Original Message----- From: Lorrie Epling [mailto:lepling@medsfgh.ucsf.edu] Sent: Tuesday, August 06, 2002 10:17 AM To: cyto-inbox Subject: query on LPAs: replacing tritiated thymidine with flow Dear "Flow"ers, Any stories of success or failure with replacing lymphocyte proliferation assays using tritiated thymidine with a flow based assay? Thanks, Lorrie Epling Laboratory Supervisor UCSF/SFGH Core Immunology Lab General Clinical Research Center Phone 415-476-5279 Fax 415-206-8200
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