Dear Flow-ers:
Thanks for the responses about alternatives to radioactive NK assays.
Has anyone performed an NK assay using PBMCs that had been frozen, then
thawed?
Lorrie Epling
Laboratory Supervisor
UCSF/SFGH Core Immunology Lab
General Clinical Research Center
Phone 415-476-5279
Fax 415-206-8200
In a message dated 7/3/02 1:23:19 PM, lxl7@CDC.GOV writes:
<< 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
>>
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