Real-time cytotoxicity assay (xCELLigence) was carried out to analyze the cytotoxicity of the CD8+ CAR-T cells as previously described19 (link) and using an Effector:Tumor cell ratio of 1:1). All experiments were performed in duplicate.
CAR-T cell cytotoxicity was also measured by flow cytometry. 6×105 CAR-T cells were cocultured with PM299L-EDAhigh PM299L-EDAlow or PM299L-Thy1.1 cells for 24 hours at two different Effector:Tumor ratios (1:1 and 0.2:1). Then, cells were washed and incubated with a fluorochrome-conjugated antibody against CD8. Perfect-Count beads (Cytognos) were added for the flow cytometric quantification of absolute cell numbers.
CAR-T cell cytotoxicity was also measured by flow cytometry. 6×105 CAR-T cells were cocultured with PM299L-EDAhigh PM299L-EDAlow or PM299L-Thy1.1 cells for 24 hours at two different Effector:Tumor ratios (1:1 and 0.2:1). Then, cells were washed and incubated with a fluorochrome-conjugated antibody against CD8. Perfect-Count beads (Cytognos) were added for the flow cytometric quantification of absolute cell numbers.
