Karim- I agree with Mark that there is no magic to the single platform assay and that many types of beads will do. We used to use glutaraldehyde-fixed chicken red blood cells: broadly fluorescent, stable for years and essentially free. The trick that makes the clinical assays work so well is the lyse/no wash protocol. As soon as a wash step is involved there is the danger of loss of cells and/or beads with no assurance that loss is proportionate. The lyse/no wash protocol leaves a lot of debris and requires that one of the fluorescence channels be used for CD45 (if it is important to get a "total leukocyte" denominator). If you wish to get a simple nucleated cell count, PI staining of permeabilized cells, plus the bead du jour will do the trick. One further caveat: In the single platform assay, pipetting accuracy (of beads and cells) is everything. Albert Can anyone recommend a microsphere kit for performing simple cell counts on numerous samples using the FACS? BD's Truecount beads are only sold along with a MAb. Our cells do not have to be labelled. Any simpe alternative? (NB: our good ol' Coulter particle counter is dead) Tx Karim
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