α cd3 pe

Cell suspensions were stained for FACS analysis with the following antibodies: α-CD3-PE (BD Biosciences), α-CD8-BV605 (BD Biosciences), α-CD45-PercP (BD Biosciences), α-CD45-PE (BD Biosciences), α-CD11b-FITC (BioLegend), α-CD11c-FITC (BioLegend), α-CTLA4-PE (BD Biosciences), α-granzyme B-Alexa fluor 647 (BD Biosciences), α-IFNγ-APC (BD Biosciences), α-KI67-BV421 (BD Biosciences), α-TIM3-PE (eBioscience), α-TIGIT-PE (BD Biosciences), α- TNFα-PE (BD Biosciences), α-IL17r-PE (BD Biosciences), and the corresponding isotype control (mouse IgG1, Southern Biotech). The live/dead fixable near-infrared dye (Invitrogen) was used to exclude dead cells. Intracellular fixation and permeabilization buffers from eBioscience were used for cytokine staining.
Acquisition was performed on FACS BD Fortessa and the dedicated software CellQuest (BD Biosciences). Data was analyzed with FlowJo 7.5.5 software (TreeStar Inc).

For survival and class switch recombination experiments, mouse spleens of RD/RDF WT and KO mice were isolated and single B cell suspensions of the whole tissue were prepared after MACS depletion as described above. Activated B cells were stained with α-B220-BV785 (103246; BioLegend), α-IgG1-FITC (553443; BD) and TO-PRO3 (Invitrogen). CSFE cultivated, activated B cells of RD mice were stained with α-B220-APC (103212; BioLegend) and DAPI (Sigma).
Mouse spleens of RDM/RDFM WT and KO mice were isolated and cell suspensions of the whole tissue were prepared. Cells were stained with α-CD3-PE (553064; BD), α-B220-BV785, α-B220-PE (553090, BD Biosciences), α-CD19-BV421 (115,549; BioLegend), α-IgM-FITC (553408; BD Biosciences), α-CD138-Pe-Cy7 (142514; BioLegend), Sca-1 (or Ly-6A7E; 108139; BioLegend), and TO-PRO3 or DAPI . Flow cytometry was performed on a LSR Fortessa (BD Biosciences) and analysed in FlowJo (FlowJo, LLC).