Pe cy7 conjugated anti cd80

Expression of cell surface molecules and intracellular cytokine were evaluated using flow cytometry. A minimum of 10,000 events was acquired using a Beckman-Coulter (Brea, CA, USA) CyAn ADP flow cytometer and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-human Vimentin, phycoerythrin (PE)-conjugated anti-human Tim-3(Biolegend), PE-conjugated anti-human PDL-1 or active Caspase-3, allophycocyanin (APC)-conjugated anti-human PDL-2, (BD, San Jose, CA, USA), FITC-conjugated anti-IFN-γ or IL-1β, PE-conjugated anti–Vimentin, PE/CY7-conjugated anti-CD80 or IL-10 or TNF-α, APC-conjugated anti-CD86 or IL-5 or IL-13, Brilliant Violet 421-conjugated anti–IL-2 or –IL-4 (Biolegend) and anti-human/mouse IRF4 eFluor® 660 (eBioscience, San Diego, CA, USA). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm Kit (Biolegend). Cell fluorescence was measured using a Beckman-Coulter CyAn ADP flow cytometer and analyzed with FlowJo software (Tree Star). Murine IgGs of the same isotype were used as negative controls.

A total of 2.5 × 10⁶ splenocytes were mixed with purified MARV GP (10 mg/L, from the 293F expression system) were seeded into 12-well plates. After 72 h of culture, cells were collected by centrifugation and resuspended in PBS containing 2% FBS and 0.1% NaN₃. Non-specific Fc receptors were blocked with 1 μg of anti-mouse CD16/CD32 antibody (BioLegend, 101301) per sample. Each sample was stained with 1 μg of APC-Cy7 conjugated anti-CD3e (ThermoFisher Scientific, 47-0031-82), 0.25 μg FITC-conjugated anti-CD4 (ThermoFisher Scientific, 11-0041-82), 0.25 μg PE-conjugated anti-CD8a (BioLegend, 100707), 0.5 μg APC-eFluor780 conjugated anti-B220/CD45R (ThermoFisher Scientific, 47-0452-82), 0.25 μg PE-conjugated anti-CD138 (BioLegend, 142504), 0.5 μg PE-Cy7 conjugated anti-CD80 (BioLegend, 104734), and 0.06 μg APC conjugated anti-CD62L antibody (ThermoFisher Scientific, 17-0621-82) for 40 min on ice. The fluorescence signal was recorded using a flow cytometer (BECKMAN COULTER, CytoFLEX).