Cd23 fitc

Flow cytometry was used to assess the following cell lineages: B cells (CD3⁻CD19⁺), plasma cells (CD3⁻CD138⁺), FCER2⁺ B cells, and FCER2⁻ B cells. PBMCs from the above patients were isolated from whole blood using density gradient centrifugation in Ficoll/Hypaque for 20 minutes at 400 × g with the brake-off. The PBMCs were washed and stained with the indicated fluorescence-labeled antihuman antibodies for 30 min on ice and protected from light. The cells were then washed and fixed in 2% formaldehyde. The samples were acquired using a FACSCanto cytometer and analyzed using FlowJo v.10 (Treestar, Seattle, California, USA). The corresponding isotype controls were used to determine the negative gate. Antibodies used in this study included CD23-FITC (#338505), CD19-PE (#302207), CD3-APC (#300311), and CD138-APC-Fire^TM 750 (#352315, BioLegend, San Diego, California, USA).

Single-cell suspensions from spleens, bone marrow, or peripheral lymph nodes were blocked for 30 min using FBS. Cell-surface staining was achieved by incubating cells at 4°C for 30 min with fluorescence-conjugated anti–mouse antibodies (clone; source): XBP1s–Alexa Fluor 647 (Q3-695; BD), XBP1s-phycoerythrin (PE; Q3-695; BD), B220–Alexa Fluor 488 (RA3-6B2; BioLegend), B220-BV605 (RA3-6B2; BioLegend), CD43-PE (eBioR2/60; eBioscience), CD19–Alexa Fluor 647 (6D5; BioLegend), IgM-PE-Cy7 (RMM-1; BioLegend), IgD-FITC (11-26c.2a; BioLegend), GL7-PE (GL7; BioLegend), AA4.1-PE-Cy7 (AA4.1; BioLegend), CD1d-PerCP-Cy5.5 (1B1; BioLegend), CD23-FITC (B3B4; BioLegend), CD3-APC-Cy7 (145-2C11; BioLegend), CD4-BV605 (RM4-5; BioLegend), CD8α-PE-Cy7 (53–6.7; BioLegend), and CD138-PE (281–2; BioLegend). Viability staining was accomplished using DAPI exclusion during acquisition. Acquisition of B, T, and dendritic cell populations was performed on an LSRII cytometer (BD) harboring a custom configuration for the Wistar Institute. Cytometry data were analyzed using FlowJo software (7.6.1; Tree Star Inc.).

WT and mFICD^−/− mice aged 6–8 weeks were euthanized by CO₂ asphyxiation followed by cervical dislocation. Spleen, bone marrow, and thymus tissues were extracted and homogenized in PBE buffer (PBS +0.5% BSA and 1 mM EDTA). Red blood cells were lysed using ACK lysis buffer (Gibco), and cells were resuspended in PBE for surface staining with the following antibodies (clone;source) for 30 min at 4˚C: B220 PerCP-Cy5.5, CD43 APC, CD19 Pacific Blue, IgM FITC, IgD APC-Cy7, CD21 PerCP-Cy5.5, CD23 FITC, IgM APC, CD44 FITC, CD25 PE-Cy7, CD4 APC, CD8α Pacific Blue (53–6.7;BioLegend), CD3 PerCP-Cy5.5 (145-2C11;BD), B220 APC-Cy7. All samples were blocked using Fc-Block (BD Bioscience). Acquisition of B and T cell populations was performed on an LSRFortessa cytometer (BD) instrument and analyzed with the FlowJo software package (Tree-Star).