Dear Erik, variations in optimal T:E ratio depend mostly on enrichment of effector T cells that is achieved in your experiments. If you deal with cloned CTLs, you will see the most stable results in restricted range of T:E ratios (may be from 10:1 to 1:10). If you deal with polyclonal populations stimulated in vitro, optimal ratio can vary significantly from one experiment to another. In this case you will need to use more wide range of ratios (may be from 1:10 to 1:300). As a rule, CTLs isolated ex vivo after immunization will require more than 100 effector cells per 1 target. I hope, you well know that this sophisticated assay implies the putting of a set of controls to estimate spontaneous and maximal destruction of target cells (in Cr-51 or similar asays), as well as controls to estmate specificity of CTLs. Good luck in your experiments!

Hi Dmitry,

thanks for your answer. I have indeed MACS purified CD8 cells from mice. Given the high ratios needed then, I may have to run a few tests on the side to see what the best target seeding density is on non-96 well plates as I doubt I can keep 10^6 cells/well happy and alive for 3-6 days in a 96 well plate..

But definitely good to know the general outlines to test. Thanks again!

Dear Erik, only small part of ex vivo isolated CD8+ cells wil be specific CTL and significant part of them will die in vitro. Really, you will need to collect cells from 6-12 wells of 24 well plate to perform conventional CTL Cr-51 assay. In the past, to minimize "cell consumption", I used in vitro modification of Winn's test. To do it, effectors were seeded at the concentration allowing their further welfare for several days. Then, I added small numbers of tumor targets (for example, 100 or 1000 of target cells per well) and allowed them to grow during several days. Then, MTT was added for 2-4 h to determine the ratio, at which tumor cells are fully destroyed (100% lysis), The presence of effector cells practically does not affect results, because after several days in vitro they transform MTT minimally.

Yes, that would be a good approach for a normal assay, but I'm looking at caspase activation by fluorescence so I need a) targets attached to a focal plane so I can image them and not floating around and b) high enough numbers to accurately detect fluorescence. The fact that only a few t cells are specific for any given antigen is not idea, but IMO also not a big impediment as I am comparing immunized and non immunized groups and thus am only interested in a relative difference between the two.

1) If you see responses to alloantigens and study CTLs specific to allogeneic class I molecules, next approach would be helpful. Perotoneal macrophages can form very strong monolayers on polystirol flasks. If the macrophages carry respective alloantigen, they can be very good targets corresponding your needs. After short incubation with these monolayers, specific CTLs are attached, whereas unattached cells may be removed by gentle washing with warm media. This procedure allows to enrich CTLs specific to alloantigens ~ 20x. Then, you can see all subsequent stages of letal hit in vitro under microscope. However, unspecific attachment of lymphocytes to monolayers also will take place.

2) Monolayers of macrophages may be incubated with anti-CD3 antibodies. These antibodies will be attached to Fc receptor of macrophages. After this, macrophages will be targets for any effector CTLs irrespective of specificity. Subsequent steps may be analogous. Hope, these considerations will be helpful.