Anti cd27 apc clone o323

PBMCs were stained for 15 min in cell staining buffer (BioLegend) at RT in the dark using the following antibodies: anti-CD3–PerCP (clone OKT3; BioLegend), anti-CD19– Brilliant Violet 510 (clone HIB19; BioLegend), anti-CD27–APC (clone O323; BioLegend), anti-CD38–APC-Cy7 (clone HB-7; BioLegend), and anti-CD20–FITC (clone 2H7; BioLegend).
The following B cell population was sorted using a FACSAria cell sorter (BD Bioscience): CD3–CD19+CD20-CD27++CD38^high.
B cell subpopulations were sorted and collected into TRI Reagent solution (Sigma Aldrich) and frozen at −80°C.

Cryopreserved PBMCs were thawed at 37°C and resuspended in RPMI 10% FBS, washed with PBS 1X and fixed with 1% paraformaldehyde for 20 min at room temperature. After washed with FACS solution (PBS 1%, sodium azide 0.05% and 2% FBS), cells were incubated for 30 min at 4°C with the following antibody cocktail: anti-CD3 PerCP/Cy5.5 (clone SKY; Biolegend; 344808), anti-CD19 FITC (clone HIB19; Biolegend; 302206), anti-CD20 PE Cy7 (clone 2H7; Biolegend; 302312), anti-CD27 APC (clone O323; Biolegend;356410), and anti-CD38 PE (clone HIT2; Biolegend; 980302). Flow cytometry analysis was performed in a FACS Aria II (BD Biosciences, San Jose, CA, USA). Doublets and CD3+ events were gated out. Plasmablasts were defined as CD3⁻/CD19⁺/CD20⁻/CD27+/CD38⁺. 500-1000 plasmablasts were acquired per sample. Analysis was performed using Flowjo software (TreeStar).