Extended Data Fig. 4: Schematics of workflow of experiments for Fig. 2.
a. Schematics of the workflow used in Fig. 2c, d for detecting CAR endocytosis. Surface and endocytic CAR were first stained with primary anti-Flag-BV421 at 37 °C for 30 minutes, followed by a secondary anti-Rat-IgG2a-AF647 stained at 4 °C for 15 minutes. The surface, endocytic CAR and CAR- populations were differentiated based on their staining patterns of the primary and secondary antibodies. b. Schematics of the workflow used in Fig. 2e to assess the endocytosis rate of CAR. Surface CAR was initially labeled with primary anti-Flag at 4 °C, followed by incubation of CAR-T cells at 37 °C for varying durations to allow endocytosis. The remaining surface-bound CAR molecules were then stained with a secondary anti-Rat-IgG2a-AF647 antibody at 4 °C. Endocytosis was quantified by the decrease in the percentage of cells stained positive for the secondary antibody. c. Schematics of the workflow used in Fig. 2f for detecting recycling CAR. Surface and endocytic CAR were first stained with primary anti-Flag at 37 °C for 30 minutes, followed by a secondary anti-Rat-IgG2a-AF647, either stained at 4 °C for 15 minutes (baseline) or at 37 °C for 30 minutes or 60 minutes. Recycling was quantified by the increase in the percentage of cells stained positive for the secondary antibody.
