Dear Allison,

You can use Propidium iodide method for flow cytometry.

Best regards,

Thank you all for your input.  I've been concerned about a flow based assay.  I've been worried that the cells might fragment and therefore not be counted by flow.  Is that a problem with that approach?

Adding to Rachel's comments regarding Allison's question - I routinely include control samples of the labelled target population, either  mixed with irrelevant "effector population" in order to assess the total input and background death of the targets.  If you use counting beads, that can provide an input standard to compare your input counts, vs the cytotoxicity assay count of target cells. Target cells are chosen for their distinct (usually larger) size or phenotype in comparison to the effector population.

Another very nice cytotoxicity assay is the "in vivo" CTL assay.  They are fairly simple, but do require mice and lots of CFSE-labeled spleen cells to act as targets.  Control naive mice are an important control, and the inclusion of an internal target loading control - which consists of differentially labeled non-target cells, which are mixed 1:1 with the labeled antigen specific (or peptide pulsed) targets.  This mixture is injected I.V. into either the tail vein or eye sinus of your pre-immunized test mice.  Harvest their spleen any where from 6 hr - 24 hrs later, then analyze the spleen cells  of each test animal and controls to quantify the number of remaining CFSE labeled cells. Gate on, and accumulate a set number of non-target cells- so all your histgrams dislplay the same number of control target cells.  Spleens containing few antigen-specific targets in comparison to input of the 1:1 mixed population in naive mice will indicate the level of antigen specific T cell activity. 

See:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159590/

or

Biomedical Research

Vol. 32 (2011) No. 2 April P 159-166

One-step simple assay to determine antigen-specific cytotoxic activities by single-color flow cytometry

Yohko Nakagawa, Eiji Watari, Masumi Shimizu  Hidemi Takahashi

or for more multiplex target approach:

http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22084/full

If your group or department has a plate reader that has can function as a time resolved fluorometer, you could use this assay: 

http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/delfia/delfia_cytotoxicity.xhtml

It's very sensitive and not very toxic

Hi Allison

An analogous method to the Cr release assay using calcein-AM was described by Neri eta l (2001) in Clinical and Diagnostic Laboratory Immunology p1131 (Title:Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants)

We have used this method with good results (we used the micro assay described in the method) to look at NK cell-mediated cytotoxicity (but you can use it for T cell assays also) - It is all done in a 96-well tray and you use a spectrofluorometer to measure the lysis.

I cannot vouch that it is "the best" alternative to radioactivity but the researchers (above) found it comparable to the Cr release assay.

cheers

Sean

HI Rachel, Sorry, I am reading these nice dialogues on CTL assays 2 years after.  

How long do you do your CD107a for? Most papers do it for 5 hours but was wondring you or anyone has tried shorter time.

Pirouz