Anti igm pe cy7 rmm 1

Bone marrow single-cell suspensions were made by flushing femurs and tibiae with PBS + 2% fetal calf serum (FCS). All cell suspensions were treated with ACK buffer for red cell lysis. For flow cytometic analysis cell suspensions were stained with the appropriate combination of the following antibodies: anti-FceRI-PE (MAR-1; BioLegend); ant-CD49b-PE-Cy7 (DX5; Biolegend); anti-CD90.2-APC (30-H12; Biolegend); anti-CD19-APC (1D3; eBioscience); anti-IgM-PE-Cy7 (RMM-1; Biolegend); anti-IgD-eFluor450 (11-26; eBioscience); anti-CD93-PE (AA4.1; Biolegend); anti-CD2-FITC (RM2-5; BD Biosciences). Dead cells were excluded with Zombie UV Fixable viability dye (BioLegend). For cell cycle analysis and Nicoletti assay cells were fixed with the FOXP3/Transcription Factor Staining Buffer Set (eBioscience) and DNA was stained with 4,6 diamidino-2-phenylindole (DAPI; BioLegend). Flow cytometry was conducted using an LSRFortessa 5-laser (325; 405; 488; 561; 632) configuration (BD Biosciences). For FACS cells were collected using a MoFlo Astrios (Beckman Coulter) and sorted directly into Opti-MEM+ 10% FCS Media.

For serum reactivity analysis, syringe-lysed GFP-expressing strains (RH1-1 and GT1-GFP) were fixed in 3% formaldehyde for 20 minutes, washed twice in PBS, and plated in 96 well micro-titer plates at 4x10⁵ parasites/well. The parasites were then incubated with serum from chronically infected mice, at serum concentrations ranging from 10⁻² to 10⁻⁶ diluted in FACS buffer, for 20 minutes at 37°C. Parasites were then washed with FACS buffer and placed on ice for incubation with anti-isotype detection antibodies depending on the application: anti-IgG3-BV421 (R40-82, BD Bioscience), anti-IgM-PE/Cy7 (RMM-1, BioLegend), anti-IgG1-FITC (RMG1-1, BioLegend), anti-IgG1-APC (RMG1-1, BioLegend), anti-IgG2b-FITC (RMG2b-1, BioLegend), anti-IgG2b-PE (RMG2b-1, BioLegend), anti-IgG2a-FITC (RMG2a-62, BioLegend), anti-IgG2a-PerCP/Cy5.5 (RMG2a-62, BioLegend), anti-IgM-a-PE (DS-1, BD Bioscience), and anti-IgM-b-PE (AF6-78, BD Bioscience).