Fitc anti human cd63

Immediately following the stimulation protocols above, supernatants were gently removed and stored at -20°C. Then, the cells were immediately fixed with 4% paraformaldehyde and incubated for 15 minutes at 37°C before incubating with 1ug/mL of FITC anti-human CD63, FITC anti-human CD35, or APC anti-human CD107a antibody or isotype control (Biolegend) for 30 minutes at 4°C. Cells were then run through either a FACS Calibur (Becton-Dickenson) or a MACS Quant 10 (Miltenyi Biotech) flow cytometer and analyzed using FlowJo (Becton-Dickenson). The isotype control showed a lower signal at all conditions than the anti-CD63, anti-CD35, and anti CD107a antibody conditions. For detection of myeloperoxidase (MPO), supernatants stored overnight or several days were thawed slowly on ice, and tested using the BioLegend LEGENDplex^TM Human MPO Capture Bead kit according to the manufacturer’s instructions. Samples were then run through either a FACS Calibur (Becton-Dickenson) or a MACS Quant 10 (Miltenyi Biotech) flow cytometer and analyzed using FlowJo (Becton-Dickenson).

Exosomes were resuspended in 100 ul PBS and then added 10 ul aldehyde/sulfate beads (Invitrogen, Catalog #A37304) into it, rotated at room temperature for 15 min. We added 600 ul PBS and rotated at 4°C overnight and 400 ul 1 M glycine rotated at room temperature for 30 min. Samples were spined at 12,000 rpm for 1.5 min and aspirated supernatant, resuspended precipitate in 100 ul 10% BSA, rotating at room temperature for 45 min. Specimens staining was performed for 30 min at 4°C with the following antibodies: PE Anti-Human-PD-L1 (BioLegend, Catalog #329706), Alexa Fluor® 647 anti-human CD279 (PD-1) Antibody (BioLegend, Catalog # 329910), PerCP/Cyanine 5.5 anti-human CD9 Antibody (BioLegend, Catalog #312110) and FITC anti-human CD63(BioLegend, Catalog #329906) tested in Beckman Coulter CytoFLEX (CA, USA).