Anti fc receptor mab

1 × 10⁵ CD27⁺ B-cells from healthy donors were cultured in 50% complete medium supplemented with 50% plasma from either CIR or HD with or without 2 μg/ml agonist anti-Fas mAb (CH11; MBL International, Woburn, MA). In some experiments, the TLR4-antagonist Rhodobacter sphaeroides LPS (10 μg/ml, LPS/RS; InvivoGen, San Diego, CA), blocking anti-BAFF mAb(20 μg/ml, 148725; R&D Systems, Minneapolis, MN), blocking anti-Fas mAb (10 μg/ml, SM1/23; eBioscience, San Diego, CA), blocking anti-CD40L mAb (10 μg/ml, MK13A4; Enzo Life Sciences, Farmingdale, NY). In some experiments, HD CD27+ B-cells were preincubated for 30 minutes with agonistic IgG/A/M (20 μg/ml, Jackson Immunolabs, Kennett Square PA) or anti-Fc receptor mAb (BioLegend, San Diego, CA). For neutralizing circulating Immunoglobulin (Ig) in CIR plasma, circulating Ig were removed by protein A/G (Spherotech, Lake Forest, IL) before co-cultured with CD27⁺ B-cells. After 18 hours, cells were then washed in PBS and resuspended in Annexin-V binding buffer with Annexin-V-FITC and PI (BioLegend, San Diego, CA).