Hi Lee, there's quite a number of articles describing cytotoxicity assays using flow cytometry to discriminate between targets (labelled with a cell tracker) and effectors and discriminate viable from dead (targets, but also effectors) by membrane integrity stains (PI/EB...). In terms of a ready to go working protocol I found the protocol of the Orpegen company very useful: they sell a "nk kit" (complete with frosen and labeled K562 targets included) but even without buying their kit, their protocol is on their website and is very informative. http://www.orpegen.com/diagnos0.htm I found the NK-activity go down very fast during 24h storage: down to 20-30% of initial value in some cases: only freshly drawn blood should be used! Some other references: NG Papadopoulos, GVZ Dedoussis, G Spanakos, AD Gritzapis, CN Baxevanis, M Papamichael. An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry. Journal of Immunological Methods 177, 1995, 101-111. AE Mattis, G Bernhardt, M Lipp, R Forster. Analysing cytotoxic T lymphocyte activity: a simple and reliable flow cytometry-based assay. J Immunol Methods, 1997: 204, 135-142. Test kit for the quantification of the cytotoxic activity of natural killer (NK) cells. Operators manual NKTEST*, Orpegen Pharma, Heidelberg, F.R.G, info@orpegen.com. I hope this helps, succes, Dirk Prof. Dirk Van Bockstaele, PhD Antwerp University Hospital Laboratory of Hematology Head of Flow Cytometry & Molecular Diagnostics Wilrijkstraat 10 B-2650 Edegem Belgium. phone: 32 3 821 3900, fax 32 3 825 1148 e-mail: dirk.van.bockstaele@uza.be > ---------- > Van: Lam, Lee[SMTP:lxl7@CDC.GOV] > Verzonden: dinsdag 25 juni 2002 17:49 > Aan: Cytometry Mailing List > Onderwerp: NK cytotoxicity assay with human whole blood > > > Dear all, > Our lab would like to use flow cytometry for the measuring natural killer > cell cytotoxicity. Does anyone have a working protocol that can share with > us? Also, could we use 24 hrs. old human whole blood instead of PBMC for > the > assay? It will be really appreciated if you can provide us with valuable > pointers. Thanks in advance. > Lee Lam > CDC > Atlanta, Georgia > Phone: 404-639-2725 > Fax: 404-639-4838 > E-mail: LXL7@CDC.GOV <mailto:LXL7@CDC.GOV> > >
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