Fig. 5

Effect of recovery media on cell viability, transfection efficiency and secretion in human CTLs. A, B. Representative flow cytometry analysis of cell viability and transfection efficiency of primary human CTLs 12 hours after electroporation with pmax-GFP. Human CTLs were allowed to recover for 12 hours post-electroporation at 32 °C either in AIM-V (middle) or our new RM (right) and compared to untransfected cells (left). A Viable cells were gated based on forward and side scatter. Cell viability was normalized to control (untransfected cells). Numbers in plots represent the percentage of viable cells in the gate. B Transfection efficiency corresponds to the percentage of GFP-positive cells relative to the percentage of viable cells in each condition. Numbers in plots represent the percentage of GFP-positive cells in the gate. C-F represent the results obtained from human CTL transfected with GzmB-pHuji. Cells were recovering from electroporation for 12 hours either in AIM-V or our new RM at 32 °C before being placed for an additional 2 hours in fresh AIM-V at 37 °C. C. Representative brightfield images (top) and corresponding GzmB-pHuji images (bottom) of the CTLs. To allow immunological synapse formation, cells were seeded on anti-CD3 coated coverslips. Displayed images were acquired 10 minutes after seeding. Scale bar: 5 μm. D, E Quantitative analysis of cell viability (D) and transfection efficiency (E) expressed as the percentage of viable cells relative to the total number of cells and the percentage of fluorescently labeled cells relative to the total number of viable cells, respectively. Data represents mean ± SEM. N = 4 independent experiments, *** p < 0.001. F Top: TIRFM image of a human CTL transfected with GzmB-pHuji on anti-CD3 coated glass coverslip showing lytic granule exocytosis. Scale bar: 1 μm. Bottom: Sequential snapshot of one exocytosis event. Upon fusion with the plasma membrane the lytic granule becomes bright as the granule lumen is neutralized through contact with the extracellular medium. Then it quickly loses its fluorescence (in less than 300 ms) as the GzmB-pHuji diffuses away. Images were acquired at 10 Hz. Time points of the displayed snapshots are indicated in seconds