7/1 List members: I'd appreciate any information regarding the availability of commercial kits for assaying cytotoxic function in T cells, analogous to the information Dirk Van Bockstaele gave for NK cells recently (below). Thanks in advance. Mark Pavlick CMRS NIAID > ---------- > > > ---------- > > From: Van Bockstaele, Dirk > > Sent: Friday, June 28, 2002 6:19 AM > > To: Cytometry Mailing List > > Subject: RE: NK cytotoxicity assay with human whole blood > > > > > > 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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