Paula Fukushima wrote: > > Hi List, > I have a question about Becton Dickinson's Trucount product. The tubes > come with no product insert so I went on their Web page > http://www.bdfacs.com/source_book/html/23_3025.shtml. I found a procedure > for absolute CD4 counts which I have tried to adapt to a cell death > assay.When calculating absolute counts, their procedure says use a test > volume which is found on the foil package. There is no test volume listed, > just the number of beads per lot. Has anyone else used this product to > determine absolute counts for tissue culture with reproducible results? > > After getting weird results we contacted BD who states that the > procedure is wrong so I guess you cannot count on the accuracy of their Web > site. I'm pretty bitter after spending lots of time and money on this. The link you gave refers to BD's CD3/19/45 tube for clinical immunophenotyping in whole blood. The procedure with Trucount however is always the same. There is a misplacement of the star in the formula. It should refer to the number of beads per test tube, which is actually the number of beads per lot in every single tube. The test volume isn't critical, after adding the cells in a volume at your choice you can dilute as you like, as the ratio of beads to cells remains constant. For tissue culture you have to be careful with Trucounts, because you must not wash cells in Trucount tubes because there is selective loss of beads during washing. So the best is to prepare everything as usual in the common tubes and then transfer an EXACT volume into the Trucount tubes. Then you can calculate using the given formula the absolute number of cells in your tube. Be careful however, this will not really tell you the number of cells that were in your culture plate! You definitely will loose cells during preparation. This is normally not a big problem regarding the percentage of cells positive for a marker or viable or anything, but may be quite of some importance when looking at absolute numbers. Hope this helps a bit Jens Fleischer
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