Pe conjugated anti cd138

The cells were analyzed on a MACSQuant Flow Cytometer (MiltenyiBiotec) using the following specific antibodies: VioletFluor^TM 450-conjugated anti-B220 (TONBO Biosciences), Brilliant Violet 510^TM-conjugated anti-CD4 (BioLegend), Brilliant Violet 510^TM-conjugated anti-CD8a (BioLegend), phycoerythrin (PE)-conjugated anti-CD138 (BioLegend), Alexa Fluor 647-conjugated anti-GL7 (BioLegend), PE-conjugated anti-CD62L (BD Biosciences), allophycocyanin (APC)-conjugated anti-CD44 (eBioscience), APC-conjugated anti-CD86 (TONBO Biosciences), PE-conjugated anti-CD69 (BioLegend), PE-conjugated anti-CD23 (BioLegend), Alexa Fluor 647-conjugated anti-CD21 (BD Pharmingen), PE-conjugated anti-PD-1 (TONBO Biosciences), Brilliant Violet 421^TM-conjugated anti-CXCR5 (BioLegend), Alexa Fluor 647-conjugated ICOS (BioLegend), eFour450-conjugated anti-CD127 (eBioscience), PE-conjugated anti-CD25 (TONBO Biosciences) and APC-conjugated anti-CTLA4 (TONBO Biosciences). Data analysis was conducted with FlowJo software (v7.6.5; FLOWJO, LLC).

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).