Anti cd45.1 pecy7

Mononuclear BM cells, B cells after culture or splenocytes were treated 10 minutes with FC block solution (BD Bioscience) then labelled 20 minutes on ice with combination of different anti-mouse antibodies (Abs) as indicated in legends of figures. Abs included anti-B220-eFluor660 (Ebioscience, Clone RA3-6B2), anti-IgM-Fitc (Ebioscience, Clone eB121-15F9), anti-IgG1-PE (Ebioscience, Clone m1-14D12), biotin anti-IgG2a^b (BD Bioscience, Clone 5.7), biotin anti-mouse IgG3 (BD Bioscience, Clone R40-82), anti-CD138-BrillantViolet421 (BD Bioscience, Clone 281-2), anti-CD43-PE (BD biosciences, Clone S7), anti-CD45.1-PeCy7 (BD biosciences, Clone A20) and anti-CD45.2-APC-Cy7 (BD biosciences, Clone 104). Biotinylated primary antibodies were detected by incubation of labeled cells with streptavidin-PE (BD Bioscience). For apoptosis analysis, B cells were first surface labeled for ASC detection, then washed and re-suspended in 1x Binding buffer (Ebioscience) and labeled with anti-AnnexinV-Fitc (Ebioscience). Ten minutes before flow cytometry acquisition, propidium iodide (Ebioscience) was added to all samples for detection and/or exclusion of dead cells. Cells were analyzed with a FACSFortessa or a FACSCanto (BD Bioscience) and Diva (BD Bioscience) or FLowJo (TreeStar) softwares.

Lymphocytes, splenocytes or thymocytes were subjected to Fc receptor blocking using purified anti-CD16/32 antibody (BioLegend), surface molecule staining with appropriate antibodies, and fixation and permeabilization using Foxp3/Transcription Factor Staining Buffer Set (Affymetrix eBioscience), followed by intracellular staining. Cytokine staining was carried out after stimulation with 20 ng/ml phorbol myristate acetate (PMA), 709 ng/ml ionomycin and 0.67 μl/ml GolgiStop (BD Bioscience) for 4 h. Stained cells were analyzed using LSRFortessa (BD Bioscience). The following antibodies were used in addition to those mentioned above: anti-CD25-APC (1:100, eBioscience), anti-CD45.1-PE-Cy7 (1:200, BD Bioscience), anti-Foxp3-eFluor450 (1:100, eBioscience), anti-CTLA4-PE (1:100, BD Bioscience), anti-Helios-PE (1:100, BioLegend), anti-Nrp1-biotin (1:100, R&D Systems), and anti-IFN-γ-APC (1:200, Affymetrix eBioscience). Clone and catalog numbers were as follows: anti-CD25-APC (clone PC61.5) anti-CD45.1-PE-Cy7 (clone A20), anti-Foxp3-eFluor 450 (clone FJK-16s), anti-CTLA4-PE (clone UC10-4F10-11), anti-Helios-PE (clone 22F6), anti-Nrp1-biotin (catalog BAF566), anti-IFN-γ (clone XMG1.2). Dead cells were excluded from analyses by using Live/Dead Cell Viability Kit (Thermo Fisher).