Alexafluor 700 cd3

Following 6 days of culture, freshly isolated autologous NK cells were added to Mϕs at a ratio of 1:1. Cells were centrifuged at 300 g for 3 min and incubated for 16 h at 37°C with 5% CO₂. Following stimulation by Mϕs, NK cells were removed by pipetting, and incubated ± K562 cells (1:1 ratio) with an antibody against the degranulation marker CD107a (BD Biosciences 328620), GolgiStop and GolgiPlug transport inhibitor for 6 h at 37°C with 5% CO₂. NK cell degranulation and interferon gamma (BioLegend, 502509) production was examined by flow cytometry, identifying live NK cell populations using Zombie Aqua viability stain (BioLegend 423101), APC-CY7 CD19 (BioLegend, 302218), Alexafluor 700 CD3 (BioLegend, 300424), PE-CY7 CD56 (Becton Dickinson, 335791), and BUV395 CD16 (Becton Dickinson, 563785).

Spleen from immunized and control animals were analyzed for B cell and TFH responses. 1–1.5 × 10⁶ cells were first labeled with the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA) and then resuspended in BD Fc Block (clone 2.4G2) for 5 min at room temperature prior to staining with a surface-stain cocktail containing the following antibodies purchased from BioLegend® (San Diego, CA, USA): APC/ Cy7 CD45, Clone: 30-F11; Alexa Fluor 700 CD3, Clone:17A2; APC CD4, Clone: RM4-5; Brilliant Violet 510 CD19, Clone: 6D5; PE/Dazzle 594 IgM, Clone: RMM-1; FITC CD21, Clone: 7E9; PE CD23, B3B4; PerCP-Cy5.5 CD43, Clone: 1B11, and PerCP-Cy5.5 CD93, Clone: AA4.1 (Supplementary Fig. 2).

Spleens were collected from Sh2b3^+/+ and Sh2b3^E372K/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).