Brilliant violet 421 conjugated anti cd138

Flow cytometry analysis of spleen cell suspensions was performed using the following fluorescent-labeled anti–mouse antibodies: APC-conjugated anti-B220 (BD Biosciences #553092), anti-CD138 (BD Biosciences #558626) and anti-IgM (eBioscience #17–5790-82); PE-conjugated anti-IgD (BD Biosciences #558597), anti-CD23 (eBioscience #5010271) and anti-CD95 (BD Biosciences #554258); PECy7-conjugated anti-CD21 (BioLegend #123420), anti-CD95 (BD Biosciences #557653), anti-GL7 (BD Biosciences # 561530) and anti-CD86 (BD Biosciences #560582); PEVio770-conjugated anti-B220 (Miltenyi Biotec #130–102-308); FITC-conjugated anti-CXCR4 (BD Biosciences #551967) and anti-IgG1 (ebioscience #11–4011-85); Brilliant Violet 421-conjugated anti-CD138 (BioLegend #142507); PerCP-Cy5.5-conjugated anti-GL7 (BioLegend #144610), anti-CD138 (BioLegend #142510) and anti-IgM (BD Biosciences #550881), APC-Cy7-conjugated anti-CD19 (ebioscience #47–0193-82), AlexaFluor647-conjugated anti-BLIMP1 (BD Biosciences #563643). Sytox blue (ThermoFisher Scientific) or DAPI was used for the exclusion of dead cells. Data was acquired on a BD FACSCanto II (BD Biosciences) and analyzed using FlowJo v10.1 software (TreeStar).

Cells from individual diaphragms were recovered as described previously(38 (link)). After 15 min incubation with Fc block (eBioscience) and 10% normal mouse serum, cells were incubated for 15 min with PE conjugated anti-SiglecF (BD Pharmingen). Single cell suspensions of bone marrow cells and spleen cells were prepared from naïve or 90 dpi WT and ΔdblGATA mice. Cells were blocked as described above and incubated for 15 min with Brilliant Violet 421 conjugated anti-CD138 (Biolegend) and PE conjugated anti-B220 (eBioscience). Data were acquired using a Gallios flow cytometer (Beckman Coulter) and analyzed with FlowJo software (Tree Star).