Pe cy5 mouse igg1 isotype control

FAM-GreenB1 and FAM-scr-GreenB1 were diluted with binding buffer to 1 μM concentration and folded. MDA-MB-231, MCF-7, and MDA-MB-436 cells were grown in T75 flasks until greater than 80% confluence, washed with PBS twice, and dissociated with non-enzymatic cell dissociation buffer for 7–9 min. FAM-GreenB1 was added at a final concentration of 200 nM along with 20 μL of PE-Cy5 mouse anti-human CD29 antibody (559882, BD Biosciences). FAM-scr-GreenB1 was added at a final concentration of 200 nM along with 20 μL of PE-Cy5 mouse IgG1 isotype control (555750, BD Biosciences). Samples were incubated on ice for 1 h, washed twice with binding buffer, and analyzed using an Amnis FlowSight imaging flow cytometer. Spearman correlation between FAM-GreenB1 and PE-Cy5 mouse anti-human CD29 antibody fluorescence intensities was calculated using Prism 9.3.1 (GraphPad).

For western blot imaging, mouse anti-granulysin (Santa Cruz, Dallas, TX; clone F-9), mouse anti-granzyme B (Biolegend, San Diego, CA), mouse anti human granzyme H clone: 4G5, a generous gift from Dr. Joseph Trapani (Peter MacCallum Cancer Centre, Melbourne, Australia), mouse anti human perforin clone: δG9, secondary antibodies goat anti-mouse IgG infrared dye 800 (Licor, Lincoln, NE), and goat anti-rabbit IgG infrared dye 700DX (Rockland, Limerick, PA) were used for imaging. For flow cytometry experiments, cells were labeled with Alexa Fluor 488 mouse anti-granulysin, clone: RB1 (BD Pharmigen, San Diego, CA), mouse anti granzyme A, clone cb9d (Biolegend), FITC mouse anti granzyme B, clone GB11 (Biolegend), PE-Cy5 mouse IgG1 isotype control (BD Biosciences), FITC mouse anti-Perforin (BD Biosciences), and FITC mouse IgG2b isotype control (BD Biosciences).