Pe labeled h 2kb siinfekl pentamer

FACS analysis was performed using 200-μL tumor or splenocyte single-cell suspensions per antibody panel. Cells were first incubated at 4°C for 30 minutes in PBS with anti-CD16/CD32 Fc block (BD Biosciences) at 1: 100 and live/death staining (Zombie Aqua, BioLegend) at 1: 1,000, or Live/Dead fixable blue dead cell stain (Invitrogen). Next, cells were washed and stained at 4°C for 30 minutes in PBS with antibodies against CD45 (clone 30-F11), B220 (clone RA3–6B2), CD3 (clone 17A2), CD8 (clone 53–6.7), MHC-I (clone AF6–88.5), PD-1 (clone RPM1–30), PD-L1 (clone 10F.9G2), NK1.1 (clone PK136), CD11b (clone M1/70), Ly6G (clone 1A8) and F4/80 (clone BM8), all from BioLegend, at 1 μg/mL. PE-labeled H-2Kb - SIINFEKL pentamer (ProImmune) was used at 2.5 μL per staining. In some sets of experiments, absolute cell numbers were calculated by adding 10 μL counting beads (1 × 10⁶ beads/mL, Spherotech) to each sample before acquisition.

200,000 or 30,000 B16/OVA melanoma cells (F1) were injected s.c. in the right flank of C57Bl/6 mice. On day 4 mice were injected i.v. with either 100 μl PBS as a control or 40 μg of exosomes. Tumour growth was monitored and mice were sacrificed when the tumour volume reached 1,000 mm³. The tumour was cut into small pieces and incubated with Collagenase/Hyaluronidase (Roche) for 30 min at 37°C. Subsequently, the tissue was passed through a 100 μm cell strainer and the single cell suspension was stained for FACS analysis using antibodies against CD45, TCR-β, CD8 (Supplementary Table S1) and PE-labeled H-2K^b/SIINFEKL pentamer (ProImmune).