We've given up using 51Cr assays since it's become obvious that primary tumour cells (as opposed to cell lines) don't always label uniformly with chromium thus losing the direct relationship between Cr-release and percent lysis. We've found the PKH-26/PI method to be VERY reproducible (typical inter assay cv's of <10% and intra-assay cv's of ~4%). We use a slight modification of the method published in "Hatam L, Schuval S, Bonagura VR. Flow cytometric analysis of natural killer cell function as a clinical assay. Cytometry 1994; 16: 59-68." Our modified assay was first described in "Lowdell et al (1997) Bone Marrow Transpl;19:891-897." The only technical difficulty with this assay is the compensation if you have a lot of dead effector cells but we rarely have complete technical failures. A modification of this assay using PKH62 and ToPro on a two laser Calibur was published last year in J Imm Meth from the ICRF in London but I don't recall the details except that the compensation problems were nil. As for using this assay in whole blood - I can't see it working due to the autofluorescence from RBC - but how long does it take to produce a PBMC cut? Dr Mark W Lowdell Senior Lecturer in Haematology / Head of Laboratory of Cellular Therapeutics RFUCMS T - 020 7830 2183 F - 020 7794 0645
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