Reply to Rhoda Eniafe originally posted: April 7th 2006.
Subject: [Immunology] Non radioactive Cytotoxic activity assay
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Hello David, I have been try to use Calcein-AM in Cytotoxic assay, but I am
having problem with leakage and inconsistencies with the killing assay, do you
encounter this kind of problem(s) and what are the steps you take to prevent it.
Thanks Rhoda Rhoda Eniafe,
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Dear Rhoda,
I just saw your posting on Calcein-AM on the Immunology hypermail list. We
have recently tried to use a Calcein-AM assay for murine YAC-1 based NK
killing assay. Although this assay has been working very well for human NK
killing assays using PBMC + K562, we experienced almost complete dye efflux
with YAC-1, despite the fact that YAC-1 cell viability was not noticably
affected by the labelling and subsequent incubation. I have also heard that one
must be careful choosing the K562 clones, as at least one lab strain needed to be
replaced for this assay.
Possibly the differences in dye efflux between cell lines relate to the expression
of transporter proteins, which may quickly pump out the dye. This is surprising
as I would have thought that the calcein AM would be quickly immobilised
inside the cell by linkage to intracellular proteins, as I assumed Calcein-AM
acted in the same manner as CFSE and CMFDA. Nevertheless, we have tried
many different settings for this assay and can report that in our hands it does not
work for YAC-1; the 'spontaneous counts' are always as high as the 'total
counts'. Due to time limitations and the availability of other assays (see below) I
have not tried other 'cell tracker' dyes in this fluorescent-based assay.
We have always been pleased with Poly Matzinger's (thymidine-based) JAM
assay for both CTL and NK killing (Matzinger P, J Immunol Methods
1991;145:85-192). It must have required an incredible leap of intuition to
realise that a CTL assay could be based on a measurement of the loss of
apoptotic cell-derived (fragmented) DNA following washing through glass-fibre
mats. Its so simple, requires only a low amount of thymidine and it is very
sensitive.
The JAM assay works well with most murine cell lines tested, as well as splenic
conA blasts. We have found that V-bottom, NON-tissue-culture treated 96-well
plates (e.g. Greiner 651101) give best results for JAM assays as they allow
better pelleting of the cell mixtures, but thats about our only modification of the
original method. See original reference and McLellan AD et al. Blood 2002;
99: 2084-93 for more information.
Flow cytometry-based assays also give very good results and there are several
detailed in the literature. We normally use these for primary cells (e.g. dendritic
cells) which cannot be labelled with thymidine. Our version of the flow
cytometry-based CTL method uses CMFDA (see: McLellan AD et al. Blood
2002; 99: 2084-93, or method available on request).
I hope this helps. Perhaps other users will have additional comments on the
Calcein-AM based assay and other CTL and NK-killing assays.
Regards,
Alex.
--
Alexander D. McLellan, PhD
Senior Lecturer
Dept. Microbiology & Immunology
University of Otago
PO Box 56
720 Cumberland St.
Dunedin
Tel: +64 (3) 479-7728 (office) +64 (3) 479-7147 (lab)
Fax +64 (3) 479-8540
