Anti mouse pd l1 pe

All peptides (Table S1) used in this study were synthesized by GL Biochem (Shanghai, China) and used as supplied without further purification. The oligonucleotides (Table S1) were synthesized and purified by Sangon Biotech Co. (Shanghai, China). Iron(II) chloride tetrahydrate (FeCl₂·4H₂O) was obtained from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from Gibco (CA, USA). LysoTracker Green DND-26 and Hoechst 33342 were purchased from Invitrogen. Anti-mouse CD3, anti-mouse PD-1-FITC, anti-mouse PD-L1-PE, anti-mouse SIINFEKL/H-2K^b-PE monoclonal antibodies were acquired from eBioscience. Anti-mouse CD8a-PE, anti-mouse CD11c-FITC, anti-mouse CD80-PE, anti-mouse CD86-APC antibodies were purchased from Biolegend. OVA tetramer-APC was purchased from MBL. The ELISA kits for TNF-α, IL-6 and IL-12 analysis were purchased from Thermo Fisher Scientific. Anti-mouse PD-1 antibody was acquired from BioXcell. The mice used in this study were obtained from Charles River (Beijing, China).

The following monoclonal antibodies (mAbs) used: anti-rat-Nrp-PE, anti-mouse-PD-L1-PE (eBioscience, San Diego, CA), anti-mouse/rat CD28-PE (Santa Cruz, CA), anti-rat CD4-FITC, anti-rat CD25-PE, anti-rat Foxp3-PE-cyanine 5 (eBioscience, San Diego, CA, USA). After membrane and intracellular staining, cells were analyzed on FACS CantoII using the BD FACSDiva v8.0 software.

Tumor cells were treated in vitro for 18–72 h ± 10–100 ng/ml IFNγ and ±1 μM ruxolitinib (#R-6688; LC Laboratories). The cells were trypsinized or scraped, washed with PBS, and resuspended in an antibody solution. Flow cytometry was performed on the Yeti or Gallios instruments and analyzed using Kaluza software as part of the University of Colorado Cancer Center Flow Cytometry Core. Antibodies and reagents used were as follows: Foxp3 Staining Buffer Set, eBioscience #00-5523-00; Anti-Mouse PD-L1-PE, eBioscience #12-5982-81 (1:200); Ghost 510 Viability Dye, Tonbo Biosciences #13-0870-T100 (1:200); Aqua Viability, Thermo Fisher Scientific #L34957 (1:200); V500 Rat anti-mouse CD4, BD #560782; Anti-Mouse PerCP I-A/I-E (MHCII), BioLegend #107624 (1:200); MHCI (H2-D), eBioscience #17-5998-80 (1:200); MHCI (H2-K), eBioscience #17-5958-80 (1:200); VersaComp Antibody Capture Bead Kit, Beckman Coulter #B22804; and Murine Fc Block, eBioscience #14-0161-86 (1:100).