Anti human cd27 apc m t271

To analyze the distribution of peripheral blood naïve and memory B cell subsets (NMOSD n = 12, MS n = 15, and HC n = 6) and to evaluate their CD180 expression (NMOSD n = 9, MS n = 7, and HC n = 5) by flow cytometry, four-color analysis was conducted using the combination of anti-human CD19-FITC (4G7, BD Biosciences Pharmingen, San Diego, CA, USA), anti-human CD27-APC (M-T271, BD Biosciences Pharmingen, San Diego, CA, USA), anti-human IgD-PerCP (IA6-2, BioLegend, San Diego, CA, USA), and anti-CD180-PE (G28-8, Becton Dickinson, Franklin Lakes, NJ, USA) antibodies, following the manufacturer's instructions. Briefly, peripheral blood samples were incubated with antibodies for 20 min. After hemolysis, cells were washed in phosphate-buffered saline (PBS) and fixed with FACSFix (0.5% PFA in PBS). Fluorescence of labeled cells was recorded using BD FACSCalibur (BD Biosciences Pharmingen, San Diego, CA, USA) and analyzed with FCS Express 6 software (De Novo Software, Pasadena, CA, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Paque Plus density gradient centrifugation of peripheral blood samples (NMOSD n = 5, MS n = 5, and HC n = 5). PBMCs were washed twice in PBS and incubated with anti-human CD19-FITC (4G7, BD Biosciences Pharmingen, San Diego, CA, USA) and anti-human CD27-APC (M-T271, BD Biosciences Pharmingen, San Diego, CA, USA) antibodies for 30 min at 4°C, following the manufacturer's instructions. After the incubation period, samples were washed twice in PBS and taken up in an in-house buffer solution (containing PBS 1x, 0.5% BSA, and 0.75% EDTA) and filtered through a cell strainer cap into Falcon polystyrene tubes under sterile conditions. Separation of naïve (CD19⁺CD27⁻) and memory (CD19⁺CD27⁺) B cells was performed using the S3e Cell Sorter (Life Science Research/Bio-Rad, Hercules, CA, USA). The purity of naïve and memory B cell populations was checked using the BD FACSCalibur flow cytometer.