Cd19 apc

CD19⁺ B cells were purified from peripheral blood, lymph node or tonsil as in [18] (link). B-cell subsets isolated: centroblast CD10⁺CD44^loCXCR4⁺ and centrocyte CD10⁺CD44^loCXCR4⁻[18] (link), naive CD27⁻CD5⁻ and memory CD27⁺CD5⁻[49] populations. Specifically, CD19+ B cells were purified from lymph node by mechanical dissociation and flushing of the tissue with sort buffer (PBS, 1% FBS, 2 mM EDTA), followed by two rounds of red blood cell lysis (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 10 minutes each. Peripheral blood was subjected to at least four rounds of red blood cell lysis. CD19+ B cells were then purified from PBMCs via an EasySep CD19+ Positive Selection Kit (StemCell Technologies cat. 17854) or using CD19 MicroBeads (Miltenyi Biotec cat. 130-050-301) per the manufacturer's instructions to >90% purity as assessed by flow cytometry (CD19-APC, Miltenyi Biotec cat. 130-110-351). Tonsillar tissues were mechanically disrupted and subjected to red blood cell lysis. B cell subsets were isolated by CD19 MicroBeads and fluorescence activated cell sorting to collect CD10+CD44loCXCR4+ (Centroblast), CD10+CD44loCXCR4− (Centrocyte), CD27-CD5- (Naive), and CD27+CD5- (Memory) populations. Due to yield constraints, some samples were not subjected to all assays.

MACSQuant Analyzer, MACSQuant Analyzer VYB (Miltenyi Biotec), or FACSCanto, LSRII (Becton Dickinson, NJ) was used to analyze cell populations by flow cytometry. The following antibody and protein conjugates were used: CD3-PE, CD3-APC-Vio770, CD4-APC, CD4-VioGreen, CD8-APC-Vio770, CD8-VioGreen, CD14-APC, CD16-PE, CD19-APC, CD20-PerCP-Vio770, CD20-PE-Vio770, CD34-APC, CD34-PE, CD45-VioBlue, CD45-VioGreen, CD45RO-PE-Vio770, CD56-PE, CD62L-VioBlue, CD95-APC, CD45-VioBlue (mouse) (all from Miltenyi Biotec); ErbB2-Fc fusion protein (R&D Systems), anti-human-IgG (Fc gamma-specific) PE (eBioscience). 7AAD staining was used for dead cell exclusion and Violet Cell Trace (Thermo Fisher) for SupT1 cell tracking.