Joy, For sorting purposes you will not be able to use the CD3/8/4 set from BD unless the CD4 is a PerCP-Cy5.5 conjugate, the regular CD4 PerCP does not show up on the sorter due to the high laser power (unless you are using a FACSort). The Coulter reagents should work if you have the correct filters. Some filter sets do not work well in differentiating the PE/ECD/PC5 combination-and the CD45 is probably unneccesary. Here is what you need to do in a nutshell: gate on a CD3 vs. SSC to get a popluation of CD3+SSC(low) cells (T-cells), you can also gate on FSC vs SSC in addition to clean it up a little more. Using that gate look at CD4vsCD8 and you should be able to pick out the populations of choice. Your logical gating strategy for the sorter will be the same as it is on an analyzer. Gate on CD3+/SSC(low)/CD4+ cells to sort one way and CD3+/SSC(low)/CD8+ cells to sort the other way. Unless you have a MoFlo 4-way sorter you will have to do two separate sorts since you want three populations. The third population will be a gating strategy to isolate CD3+/SSC(low)/CD4-CD8- cells. For fluorochromes, I would suggest FITC/PE/PE-CY5 or FITC/PE/PerCP-Cy5.5. Brian Newsom Technical Application Specialist BD/Pharmingen -----Original Message----- From: Joy Mundy To: cyto-inbox Sent: 4/13/00 7:45 PM Subject: Sorting CD4+CD3+ cells Hi everybody, I am passing the following message on behalf of a researcher in our HIV research labs. In our lab we are familiar with the analysis of T cell subsets, currently using the Coulter tetraCHROME reagent. However this researcher is interested in cell sorting and how to set up the BD flow sorter to get the cells of interest. As a consequence I am of no use whatsoever. The antibodies that we currently have at our disposal are the Coulter tetraCHROME CD45 FITC/CD4 PE/CD8 ECD/CD3 PC5 and BD CD3/CD8/CD4 together with the BD Tritest control. Analysing the cells are no problem it's just how th set up the sorter to get the correct cells out! This is the problem; Basically what I want to do is as follows Take 40mls patient blood, ficoll separation. Stain PBMCs in such a way that I can put them thru a cell sorter and collect as many cells as possible as need to extract DNA from the following , to then do a series of PCR reactions to determine integrated HIV DNA. Interested in CD4+ lymphocytes (CD3+/CD4+), CD8+ and the small number of double negative cells (cd4-/CD8-) as these are possibly CD4+ cells that have down regulated their receptor post HIV infection. I guess the ideal scenario is to pass the cells thru and collect all CD3+ cells and then the rest. Then pass thru these CD3+ cells and differentiate between the 4s and the 8s???? Thanks any suggestions welcome ! Joy Mundy Division of Human Immunology I.M.V.S. PO Box 14 Rundle Mall PO Adelaide 5000 Australia joy.mundy@imvs.sa.gov.au Phone (08) 8222-3476 Fax (08) 8232-4092
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