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Antibodies and flow cytometry

SW Simon N. Willis
JT Julie Tellier
YL Yang Liao
ST Stephanie Trezise
AL Amanda Light
KO Kristy O’Donnell
LG Lee Ann Garrett-Sinha
WS Wei Shi
DT David M. Tarlinton
SN Stephen L. Nutt
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Single-cell suspensions were stained with mAb against mouse B220 (RA3-6B2; BD Pharmingen; 1 : 200), IgM (331.12; 1 : 800), IgD (1126 C; 1 : 800), Gr-1 (RB6-8C5; 1 : 800), CD21 (7G6; 1 : 2000), CD23 (B3B4; Biolegend; 1 : 200), IgG1 (X56; BD Pharmingen; 1 : 300), IgG3 (pooled antisera; Southern Biotech; 1 : 250), CD138 (281-2; BD Pharmingen; 1 : 600), FcgR (2.4G2; 1 : 10), BAFF-R (7H22-E16; BD Biosciences; 1 : 100), CD40 (11-0402-86; eBioscience; 1 : 200) and CD98 (RL388; Biolegend; 1 :500). Cells were analyzed on FACSCanto flow cytometers and cell sorting was carried out using FACSDiVa or Aria flow cytometers (BD Biosciences). Intracellular staining for BCL6 (7D1) was performed using the FoxP3 staining buffer set (eBiosciences). All Abs were produced in-house unless otherwise indicated. Antigen-specific B cells were identified by binding NP coupled to phycoerythrin.

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