Violettrace

Flow cytometry-based cytotoxicity assays were performed, as described in detail previously.^{6 (link)} In short, CAR-KHYG-1 or target cells (malignant cell lines or primary bone marrow mononuclear cells [BMNC] from MM patients) were stained with VioletTrace (Thermo Fisher) for 20 minutes in the dark at 20°C, to discriminate effector and target cells. After washing away unbound dye, the cells were co-incubated at indicated effector to target (E:T) ratios in 96 well round-bottom plates for 18–24 hours (37°C, 5% CO₂). Post incubation, cells were stained with appropriate markers to further distinguish the MM cells and other cells when necessary. Flow-count fluorophores (Beckman 7547053) were added, and the cells were analyzed with flow cytometry as described in the previous section. Viable single cells were enumerated by excluding dead cells, doublet events and appropriate VioletTrace staining. The percentage of cell lysis was calculated as followed: % lysis cells = (1–[absolute number of viable target cells in treated wells/absolute number of viable target cells in untreated wells]) × 100%. The lysis was only calculated when the target cell population contained at least 500 viable cells in the untreated control. An example of the gating strategy employed can be found in Figure S3 (http://links.lww.com/HS/A161).