Anti human pd 1 fitc

Peripheral blood mononuclear cells were stimulated with either a 1, 10, or 25 μg/mL suspension of OMVs from both strains or a 5 μg phytohemagglutinin (PHA) solution for 12, 24, and 48 h. Plates were incubated at 37°C under 5% CO₂. Monoclonal antibodies (mAbs) coupled to the following fluorochromes were used: anti-human CD91-eFluor 660 (eBioscience), anti-human CD3-APC (BD Pharmingen^TM), anti-human CD19-PE-Cy7 (BD Biosciences), anti-human PD-L1-PE (BioLegend), anti-human PD-1-FITC (BioLegend), anti-human CD86-PE (BioLegend), anti-human CD69-TRI-COLOR (Molecular Probes). After stimulation, 100 μL of supernatant were collected from wells and stored at −70°C until usage. Then, PBMCs were collected and washed with FACS buffer, followed by incubation with diluted mAbs in FACS buffer for 1 h at 4°C (protecting the reaction from light). Finally, cells were washed with FACS buffer, fixed with PBS-PFA (Paraformaldehyde) 1%, and resuspended in 400 μL of FACS buffer. Samples were analyzed in LSRFortessa^TM cytometer (BD Biosciences), providing a total number of 30,000 events, and the resulting data was analyzed with the aid of the FlowJo^® software.