Below are the responses to my query of NK cytotoxicity assay and thank you to all who responded. If you don't see your response here, please let me know and I will make another posting in a later time. Thank you again! Lee Charles --You could try a re-directed killing assay. In mice (B6) we use a FcR+ cell line to present mAbs that crosslink NK activating receptors. Protocol is in Curr Prot Immunology Gold Book. I'm not sure if a comparable assay is set up in humans... Gerhard --Have a look at the Orpegen.de website. They have a very good protocol to their test. Only that you have to make up your own target cells. Dave --I used to run NK assays using a K562 cell line. The assay was a chromium release assay (dead cells released radioactive chromium), but I don't see why it could not be adapted to use PI and run on a flow cytometer. That assay did use PBMC's though Stan --Please post responses to the list, and I'd be interested in hearing more generally about what non-radioactive assays are used in current practise. This issue has been around awhile now, LDL assay (Promega ?) seems to have both proponents & detractors.While several assays have been validated against chromium release assays, which remain the gold standard, somehow non-radioactive assays have not replaced routine use of the chromium release assay. We still use it to measure cytotoxicity. Is it a standardisation issue? Mark --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? --most of our effector cell samples are over 24 hours old (in preservative-free heparin) and kept at 21oC and our target cells are nearly always cryopreserved so I think you will be OK. Also, we find that the flow assay is more sensitive than the 51Cr so we routinely use a 10:1 E:T ratio Dirk --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 frozen 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 <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 <mailto:info@orpegen.com> . Derek-- We have published a method recently using PKH-26 to label targets and assess cytotoxicity with TO-PRO-3 (this requires a red source for excitation). The advantage is that there is no spectral overlap. The relevant references are: Lee-MacAry et al: Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide. J Imm Methods (2001) 252, 83-92 Wilkinson et al. Antibody-dependent cell-mediated cytotoxicity: a flow cytometry-based assay using fluorophores. J Immunol Methods (2001) 258,183-191 Bill-- We have used a NK/cytolytic T cell assay from Oncoimmunin, Inc. (the CyToxiLux kit) to measure human NK activity. The assay uses a fluorogenic caspase 6 substrate (similar to their PhiPhiLux caspase 3 substrate) that is loaded immediately after target:effector coincubation - target cells are tagged prior to coincubation with a proprietary red tracking dye to distinguish them from effectors. Both probes excite at 488 nm, so you can analyze on almost any benchtop flow cytometer, gating on the tagged targets. Caspase 6 is probably one of the earliest cytolysis-associated events you can detect using current methods - it gives much higher sensitivity than 51Cr in our hands. We've used it with both peripheral blood derived NKs and NK92 cells, using K562 and Jurkat targets. Oncoimmunin's web site is at http://www.phiphilux.com <http://www.phiphilux.com/> . We have some NK data using this assay posted on our website at http <http://home.ncifcrf.gov/ccr/flowcore/cytox.htm> ://home.ncifcrf.gov/ccr/flowcore/cytox.htm <http://home.ncifcrf.gov/ccr/flowcore/cytox.htm> . Judy-- We developed our flow cytometric NK cytotoxicity assay with human whole blood and have also used the method for hamster whole blood. Journal of Immunological Methods, 166 (1993) 45-54. --Sorry for the mistake. Our method NK cytotoxicity assay does use PBMC separated by density gradient. (I was thinking of our whole blood oxidative burst.) Giovanna-- we have recently purchased the fluorogenic cytotoxicity assay from OncoImmunin (CytoxiLux) (see Nature Med. 8:185-189, 2002). I like the idea that you can combine the cytotoxicity assay with multiparametric flow cytometry and characterize the effector cells in mixed populations (such as PBLs). We haven't tried it yet, but it looks very neat, and I wouldn't be surprised if it turns out to be more sensitive than 51Cr. I'll let you know. -----Original Message----- From: Lam, Lee Sent: Tuesday, June 25, 2002 8:50 AM To: 'cytometry@flowcyt.cyto.purdue.edu' Subject: 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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