Anti igm r6 60

Lung cells were stained extracelullarly with anti-CD3 (17A2, conjugated to AF700, eBioscience), anti-CD19 (1D3, conjugated to APC, BD Bioscience), anti-IgM (R6-60.2, conjugated to PerCp-Cy5.5, BD Bioscience), anti-IgD (11-26c.2a, conjugated to PE, BD Bioscience), anti-CD95 (Jo2, conjugated to PE-Cy7, BD Bioscience), anti-GL7 (GL7, conjugated to Fitc, BD Bioscience), and a fixable live-dead marker (conjugated to eFl506, eBioscience).
Acquisition of samples was performed on a LSR II or Fortessa cytometer equipped with FACSDiva software (BD biosciences). GC B cells were defined as CD3⁻CD19⁺IgM⁻IgD⁻CD95⁺GL7⁺. Final analysis and graphical output were performed using FlowJo software v9 (Tree Star, Inc.).

The following antibodies were used for FACS staining at 200-fold dilution except for anti-CD90 antibodies, which were diluted to 2,000-fold: eFlour450-conjugated anti-CD4 (RM4-5), anti-CD8 (53-6.7) and anti-B220 (RA3-6B2) (eBioscience, San Diego, California); BV421-conjugated anti-CD19 (6D5) (BioLegend, San Diego); FITC-conjugated anti-CD44 (IM7) (eBioscience) and anti-IgD (11-26c.2a) (BD Biosciences, San Jose, California); PE-conjugated anti-CD25 (PC61), anti-CD45.2 (104), anti-CD62L (MEL14), anti-IL-17A (eBio17B7) (eBioscience), anti-IgM (R6-60.2) and anti-IgG1 (A85-1) (BD Biosciences); PE-Cy7-conjugated anti-CD3 (145-2C11) (BioLegend), anti-CD8 and anti-CD44 (eBioscience); APC-conjugated anti-CD4, anti-CD45.1 (A20), anti-CD90.1 (HIS51), anti-CD90.2 (53-2.1), anti-interferon (IFN)-γ (XMG1.2) (eBioscience), and anti-CD19 (BioLegend); biotin-conjugated CD273 (TY25) (BioLegend); anti-Bim (Cell Signaling, Tokyo, Japan); and Alexa488-conjugated anti-rabbit IgG (Invitrogen, Tokyo). Antibodies for western blotting were as follows: anti-Bim and anti-phospho S51 eIF2α (Cell Signaling); anti-FLAG M2 affinity gel and 3xFLAG peptide (Sigma, Tokyo); and anti-eIF2α, anti-Actin, anti-PP1 and HRP-conjugated anti-cMyc (Santa Cruz, Dallas, Texas). They were used at 100-fold dilution.