FCM-NK Cells

Antony Bakke (bakkea@ccmail.ohsu.edu)
Thu, 22 Jun 95 08:31:21 PST

The original paper on NK assays by FCM is: McGinnes K, Chapman G,
Marks R, and Penny R, A fluorescence NK assay using flow cytometry,
J Immunol. Methods, 86: 7-15, 1986. These authors used
carboxyfluorescein diacetate to label the targets and Coulter
fulbright beads as an internal counting standard. This works well,
but the carboxyfluorescein does leak out of the cells after a few
hours. You begin to see the mononuclear cells become fluorescent
after about 4 hours of incubation. They are still much dimmer than
the targets, if you are using standard K562 for human of YAK for
mouse. However, if you looking at other targets, such as CLL cells,
the targets are only slightly brighter than the mononuclear cells
after 4 hours. The assay is more sensitive than the standard
chromium release, so a 3 hour incubation is sufficient to measure
killing.

After setting up this original assay, I investigated some other
dyes. 5(and-6) carboxyfluorescein diacetate, succinimidyl ester
(CFSE) from Molecular Probes (cat no C-1157; phone no (503) 465-4953
for technical assistance
or (800) 438-2209 for orders) worked better, because it covalently
links to cytosollic proteins and does not leak out of the cell
quickly. The targets retain their label for 18-24 hrs.

In addition, it is helpful to set up the assay in tubes that will
fit on your flow cytometer directly and/or to use an autoloader. I
always did each point in duplicate rather than triplicate as is
usual in the chromium release assay. This helped to keep the number
of tubes managable.

You will be measuring fluorescent cells remaining rather than
release (or dead cells) so the equation to calculate cytotoxicity
changes.

Hope this helps. If you have more questions, e-mail me.

Tony Bakke (bakkea@ohsu.edu)