I purchased Bright Glo (#E2620) for luciferase based killing assay. I co-cultured my effector T cell with target tumor cells (stably express luciferase) with different ratio in 200ul T cell media in 96 well black wall plate, and 20h later, remove 100ul media, then add Bright Glo reagent. The luminesence detected was really good.

But I do not understand why the control T cells can also lyse target tumor cell (luminescence is low). Namely, as the percentage of control T cell goes higher, the lower luminescence was detected. Definitely, there are significant difference between effector T cell and control T cell. But still, it does not make sense. The method is refer to a 2015 published paper (Rational development and characterization of humanized anti–EGFR variant III chimeric antigen receptor T cells for glioblastoma), you can find Figure3-A which is shown the luciferase-based killing assay(they also have the similar curve which show control T cell has the killing function). Is that possible, the T cells can hide the luminescence? and the more T cells in culture the lower luminescence? Or is that a kind of nonspecific signal? I really curious about the mechanism of this luciferase based killing assay.

Besides, I do have the control well which is only Tumor for minimum killing, and anther well for tumor+TritonX100 for Maximum killing.

the equation is like this:

%Specific Lysis=100%x(Spontaneous death RLU- test RLU)/(Spontaneous death RLU-Maximal Killing RLU)

Briefly, Why in control T + target tumor also shows similar killing curve with experimental cohort?

Thank you very much!

The assay result was attached