Iga fitc

Peripheral blood mononuclear cells were washed in PBS with 1% BSA (bovine serum albumin) and incubated for 30 min at 25°C with fluorescently labeled antibodies specific for B and T cells. Subsequently, samples were centrifuged and resuspended in propidium iodide (PI) solution (1 μg/ml PI and 10 μg/ml RNase A in PBS) and analyzed using BD FACS Canto II flow cytometer (BD Biosciences). The B cell panel included the following antibodies: IgA FITC, IgD PE, CD3 PerCP-Cy5.5, CD27 APC-H7, CD19 PE-Cy7, β7 APC, and CD38 BV421 from BD Biosciences. The T cell panel included the following antibodies: CXCR5 BV421, CXCR3 PE, CD4 APC-H7, CD3 FITC, CD196 APC, CD279 (PD-1) BV510, and CD45RA PE-Cy7 from BD Biosciences. Results were analyzed using FACSDiva software (BD Biosciences) and reported as MFI, reflecting the levels of cell surface antigens and relative cell count with respect to hierarchically higher cell populations (%). Cellular aggregates were eliminated using morphology parameters (FSC-A and FSC-H) (see Supplementary Figures 1, 2 for gating strategy).

Single cells were isolated and labeled with a fixable viability dye and antibodies to IgA-FITC (BD Biosciences, #553478); IgA-PE (BD Biosciences, #562141); CD69-BV786 (BD Biosciences, #56468); B220-BV605 (BD Biosciences, #563708); CD19-PerCp-Cy5.5 (BD Biosciences, #551001); CD11c-PE-CY7 (BD Biosciences, #558079); CD317 (pDCA1)-BV421 (BD Biosciences, #566431); CD268 (BAFF Receptor)-APC (eBioscience, #17-9117-42); and LIVE/DEAD™ Near IR- Dead Cell Stain Kit (Molecular Probes, #L34975). Isotype controls were used to set the photomultiplier tube (PMT) voltages. Single color controls were used for compensation. Stained cells were assayed on the LSRFortessa™ cell analyzer (BD Biosciences), and flow data were analyzed and plotted using BD FACSDiva™ (ver. 8). Singlet live cells were gated before further analysis (Supplementary Fig. 1).