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. 2018 Jan 9;8:199. doi: 10.1038/s41598-017-18606-1

Figure 7.

Figure 7

Applications of the Matador assay for measuring cytotoxicity induced by CAR-T cells, Blinatumomab and Rituximab. (A) Flow cytometry analysis of NK92MI cells stably transduced with chimeric antigen receptors targeting CD19 (FMC63) or a control CAR based on detection of co-expressed eGFP expression. (B) Raji cells stably expressing Nluc were co-cultured in a 384-well plate at an E:T ratio of 0.5:1 for 4 hours with NK92MI cells expressing the indicated CARs in 60 μl total volume. Post-incubation, luminescence was detected by the addition of 15 µl of coelenterazine-containing assay buffer directly to each well. (C) Primary human T-cells transduced with the indicated CARs were co-cultured in a 384-well plate at an E:T ratio of 10:1 for 4 hours with Raji cells stably expressing Nluc, followed by the detection of luminescence as described earlier. (D) Bar graph depicting the percentage specific lysis of Raji cells by indicated CAR-Ts. The luminescence values of cells incubated with media alone is considered as 0% (spontaneous cell death or basal) and the digitonin-induced cell death is considered as 100% (maximum cell death or total). (E) Raji cells stably expressing Gluc were treated with Blinatumomab at a concentration of 100 ng/106 cells for 30 minutes at 37 °C in PBS containing 2% FBS. Next, antibody-treated cells were washed three times in PBS-FBS, re-suspended in XVIVO-15 medium and co-incubated with primary human T cells at a E:T ratio of 20:1 for 4 hours. Luminescence was detected by the addition of coelenterazine-containing assay buffer directly to each well. (F) Raji cells stably expressing Gluc were co-cultured with peripheral blood mono nuclear cells (PBMCs) from healthy donors at an E:T ratio of 40:1 for 4 hours in the presence of indicated concentrations of Rituximab, followed by the detection of luminescence as described earlier. The values shown are mean ± SE of a representative experiment performed in triplicate for at least two times. Statistically significant difference is shown by asterisks (****) at a level of P < 0.0001.