Apc conjugated anti cd138 clone 281 2

Single-cell suspensions of the spleen and bone marrow were prepared and stained with phenotypic and functional markers to assess the germinal center (GC) reaction, T follicular helper (T_FH), and humoral response. All antibodies were purchased from BioLegend, unless stated otherwise. Monoclonal antibodies against mouse antigens used include FITC-conjugated anti-B220 (clone RA3-6B2), anti-CD8 (clone 53-6.7), anti-CD95 (clone Jo2; BD Pharmingen), PE-conjugated anti-CD4 (clone H129.19) and CXCR5 (clone L138D7), PERCP/Cy5.5-conjugated anti-CD4 (clone RMA4-4), PE/Cy7 conjugated anti-PD1 (clone RMP1-30), Alexa Fluor 700-conjugated anti-CD44 (clone IM7), APC-conjugated anti-CD138 (clone 281-2) and Brilliant Violet 510-conjugated anti-B220 (clone RA3-6B2). Alexa Fluor 594-conjugated peanut agglutinin (PNA; Invitrogen) was also used. Cells were analyzed in a BD FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, U.S.) running BD FACSDiva Software version 6.1.3 (BD Biosciences), and results analyzed using FlowJo version 10.7.1 (FlowJo LLC, Ashland, OR, USA). GC B cells were defined as B cells (B220⁺ CD4^-) expressing CD95 and PNA. T_FH cells were defined as CD4 T cells (B220^- CD4⁺ CD44⁺) expressing CXCR5, along with PD-1. These cell populations were assessed in the spleen. Long lived plasma cells (LLPCs) were characterized, in the bone marrow, as B220^-/low CD138⁺ cells.