Hey Doug and Lorrie, Doug, to speed up the analysis leg of CFSE proliferation work, you could consider using FlowJo. When we do CFSE proliferation assays (for mouse work), data analysis using FlowJo makes the whole business of getting the data into excel for analysis a breeze. The Table editorš quickly applies the same gate logic to all the different batches of samples and then produces a table that can then be opened in excel, containing all the raw data for a particular batch of samples. Copying and pasting this into a pre-defined excel worksheet makes relatively light work of things. So it might not end up taking that much longer than the work required to turn CPM from thymidine incorporation assays into pretty proliferation graphs. Joanna > 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: Cytometry Mailing List > 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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