Pe conjugated anti human cd27 abs

Mononuclear cells (PBMC) were purified by Ficoll gradient centrifugation from 20 mL of peripheral blood samples. PBMCs were stained with FITC-conjugated anti-human CD3 Abs, BV421-conjugated anti-human CD19 Abs, BV510-conjugated anti-human CD20 Abs, PE-conjugated anti-human CD27 Abs, and APC-conjugated anti-human CD38 Abs (BD Biosciences, San Jose, CA, USA). After staining, HIV-positive samples were fixed with 4% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature (RT) to eliminate live virus. Samples were analyzed at the UNMC FACS core facility. Circulating plasmablasts were identified as CD3⁻CD19^lowCD20^lowCD27^hiCD38^hi cells by FACS Aria (BD Biosciences) and sorted into 96-well PCR plates at one cell/well with 10 µL of lysis buffer (10 mM Tris–HCl pH8.0, 10 U RNasin (Promega, Madison, WI, USA). The sorted single cells were directly subjected to RT-PCR amplification or stored at −80°C for future analysis. Gating and analysis of frequencies of plasmablasts were performed using by BD FACSDIVA™ software (Figure S1 in Supplementary Material).

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples (20 mL) by density-gradient centrifugation using Ficoll-Hypaque (GE Healthcare Life Sciences). PBMCs were stained with FITC-conjugated anti-human CD3 Abs, BV421-conjugated anti-human CD19 Abs, BV510-conjugated anti-human CD20 Abs, PE-conjugated anti-human CD27 Abs, and APC-conjugated anti-human CD38 Abs (BD Biosciences). CD3⁻CD19^lowCD20^lowCD27^highCD38^high cells were identified as plasmablasts in this study. Plasmablasts were directly sorted into 96-well PCR plates at a single cell/well in 10 μL of lysis buffer (Promega) by a FACS Aria (BD Biosciences). The sorted plasmablasts were directly subjected to RT-PCR or stored at −80°C for further analysis.