Spleens were collected from Sh2b3+/+ and Sh2b3E372K/E372K mice and meshed to generate single-cell suspension, which were stained with antibody cocktail containing BV480-CD93 (BioLegend; cat # 136507), PE-CD19 (BioLegend; cat # 115508), Alexa Fluor 647-B220 (BioLegend; cat # 103226) and Alexa Fluor 700-CD3 (BioLegend; cat # 100216) antibodies, then with 7-AAD (Invitrogen; cat # A1310). The cells were sorted into live (7-AAD-) transitional (CD19+CD3-B220+CD93+) and mature B cells (CD19+CD3-B220+CD93-) on BD FACSAria Fusion and FACSAria II (BD), before cultured in complete RPMI-1640 media without/with 5 μg/mL goat anti-mouse IgM (Jackson ImmunoResearch; cat # 115-006-075) and/or recombinant murine 25 ng/ml IL-4 (Peprotech; cat # 214-14) for 16 hours at 37°C/5% CO2 before staining with fixable viability dye eFluor 780 (Invitrogen; cat # 65-0865-14) and FITC-Annexin V (BD; cat # 556419) for apoptosis analysis on a LSRFortessa X-20 flow cytometer (BD). To determine BAFF-R experssion, sorting and cell culture was performed in as described above for the apoptosis assay. Following 16-hr incubation at 37°C/5% CO2, the cultured B cells were stained with Fc block followed by fixable viability dye eFluor 780 and FITC BAFF-R monoclonal antibody (Invitrogen; cat #11-5943-81) and analyzed on a LSRFortessa X-20 flow cytometer (BD).
Zhang Y., Morris R., Brown G.J., Lorenzo A.M., Meng X., Kershaw N.J., Kiridena P., Burgio G., Gross S., Cappello J.Y., Shen Q., Wang H., Turnbull C., Lea-Henry T., Stanley M., Yu Z., Ballard F.D., Chuah A., Lee J.C., Hatch A.M., Enders A., Masters S.L., Headley A.P., Trnka P., Mallon D., Fletcher J.T., Walters G.D., Šestan M., Jelušić M., Cook M.C., Athanasopoulos V., Fulcher D.A., Babon J.J., Vinuesa C.G, & Ellyard J.I. (2024). Rare SH2B3 coding variants in lupus patients impair B cell tolerance and predispose to autoimmunity. The Journal of Experimental Medicine, 221(4), e20221080.
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