Bric126

PBMCs were plated at 5×10⁴ cells per well in a 24-well tissue-culture plate at 38.5°C for 2 hours to allow peritoneal macrophages to attach to the plate. After washing off the non-adherent cells, 2×10⁵ EGFP-labeled C1 iPS cells were then added to each well as target cells. After co-incubation of macrophages and iPS cells with or without CD47-blocking antibody (BRIC126, Santa Cruz; 1∶50) for two hours, macrophages were stained with anti-porcine monocytes antibody (Antigenix America, USA; 1∶100). Phagocytic index (PI) was calculated as the number of engulfed iPS cells per 100 iPS cells.

The following primary antibodies were used in this study: CC2C6 (Biolegend), B6H12 (BD Pharmingen) and BRIC126 (Santa Cruz Biotechnology) are α-CD47 monoclonals; Mcl-1, PARP-1 (Santa Cruz Biotechnology); NOXA (Cell Signaling); 9F10 (ITGA4), P84 (SIRPα), GAPDH, and Alexa Fluor 647-labelled F4/80 (Biolegend). Secondary antibodies used were Dylight488, Dylight633, Dylight680 and DyLight800 conjugates of goat-anti-mouse, and DyLight800 conjugated goat-anti-rabbit (all from Thermo Fisher).