Cd138 pe 281 2

Single-cell suspensions of mediastinal lymph nodes (medLNs) and bone marrow were grounded and passed through 70 µm cell strainer (BD, 22-363-548). Cells were treated with ACK buffer for 3 min and then washed and spun at 800 g for 5 min, resuspended in RPMI medium plus 2% fetal bovine serum, and stained with antibodies as described below. Cells isolated from medLNs and bone marrow were counted with trypan blue, washed, and prepared for flow cytometry analysis in PBS containing 2% FBS and 2 mM EDTA. For surface staining, cells were stained with fluorescently labeled antibodies at 4°C for 15 min in the dark, followed by Live/Dead Blue (Invitrogen, Cat# L23105) staining following the manufacturer’s instructions. Samples were acquired on an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (TreeStar, versions 9.3.2 and 10.5.3.). The list of antibodies used is as follows: B220- BV510 (RA3-6B2, Biolegend), CD138-PE (281–2, Biolegend), CD95-BV421 (Jo2, BD Bioscience), GL-7-Alexa-Fluor647 (GL7, Biolegend), CD4-APC-Cy7 (GK1.5, Biolegend), and CD38-PE-Cy7 (T10, Biolegend).

Mouse spleen and lymph nodes were mechanically dissociated through a 90-mm mesh. Red blood cells were lysed using RBC Lysis Buffer (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Single cell suspensions were resuspended in PBS or complete medium. For flow cytometry, single cell suspensions were labeled with CD19-APC (6D5), CD21-Pacific Blue (7E9), CD23-PE/Cy7 (B3B4), CD93-PerCP (AA4.1), IgD-APC/Cy7 (11-26c.2a), IgM-FITC (RMM-1), CD43-PerCP/Cy5.5 (1B11), and CD138-PE (281-2) (BioLegend, San Diego, CA, USA) for 30 min at 4°C, followed by three washes with PBS. Data were acquired on BD LSR II Flow Cytometer and analyzed using FlowJo software version 10.0 for Macintosh.