Cd25 af647

The I-A^b+ B cell lymphoma line LB27.4 was purchased from the American Type Culture Collection. The following FACS antibodies were used in this study: Fc Block, CD8a BV 421, CD45.2 BV 510, B220 BV 605, CD127 AF 488, CD45.1 PE, KLRG1 PE-Cy7, CD25 AF 647, TNF-α AF 488, and IFN-γ AF 647 (all from Biolegend). Anti-alpha-adducin (ab40760) from Abcam was used for western blots. Anti-PKC-θ (C-18), anti-alpha-adducin (H-100; used for microscopy), and anti-phospho-alpha-adducin (Ser 726) antibodies were from Santa Cruz Biotechnology. CellTrace Violet, CellTrace CFSE, and AF 568-labeled phalloidin were from ThermoFisher. Class I (257-264) and Class II (323-339) ovalbumin peptides were from AnaSpec. GolgiPlug was from BD Biosciences. Anti-CD3 (145-2C11) and anti-CD28 (37.51) were from Bio-X-Cell.

CD8⁺ T cells were stimulated with agonistic antibody coated microspheres for the indicated time, washed once and stained with CD25-AF647 [Biolegend] for 15 min at 4 °C. After one washing, samples were analyzed on FACSCantoII using FACSDiva software [BD].

Fresh human PBMCs were plated at 2.0E+05 cells/well in U-bottom plates in 100 µL of complete RPMI 1640 medium. Isotype or anti-CD47 mAbs were added at a final concentration ranging from 1 ng/mL to 100 µg/mL (50 µL/well). Polyclonal T cell activation was triggered by addition of SEB (Staphylococcal Enterotoxin B, List Biological labs; Cat. 122) at a 25 ng/mL final concentration. The plate was incubated for 3 days at 37°C, supernatants collected. Cells were stained for 20 minutes at 4°C with 80 µl of an Ab mixture containing CD4-BV421 (dilution 1:200; Biolegend; Cat. 317434), CD8-PE (dilution 1:200; Biolegend; Cat. 344706) and CD25-AF647 (dilution 1:200; Biolegend; Cat. 356128), 2x washed and analyzed on flow cytometer. IFN-γ cytokine levels were measured in the supernatants by using the proinflammatory panel 1 (human) kit (Meso Scale Discovery; Cat. K15049D) as per manufacturer’s instructions.