Re: NK cytotoxicity assay with human whole blood
From: William Telford (TelfordW@mail.nih.gov)
Date: Fri Jun 28 2002 - 23:29:50 EST
Hello Lee...
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.
We have some NK data using this assay posted on our website at
http://home.ncifcrf.gov/ccr/flowcore/cytox.htm.
Enjoy,
Bill
At 11:49 AM 6/25/2002 -0400, Lam, Lee wrote:
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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