Anti b220 bv650

For flow cytometry of immune cells, lamina propria immune cells were isolated as described.⁸³ (link) For analysis of myeloid immune cells, the cells were washed in PBS, incubated with FcR blocking antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes, and stained with anti-CD45–Pacific Blue, anti-CD3–BV650, anti-NK1.1–BV650, anti-B220–BV650, anti-CD11b–BV605, anti-CD11c–PECy7, anti-Ly6C–PerCPCy5.5, anti-F4/80–APC, anti-CD64–PE, and anti–MHC-II–AF700 (all from BioLegend, San Diego, CA) for 15–30 minutes. ZOMBI-NIR live dead stain (BioLegend) was used for discrimination between live and dead cells. For cytokine staining, the cells were incubated with ionomycin and PMA in the presence of Brefeldin A for 3.5 hours before surface staining with anti-CD25–AlexaFluor700, anti-CD3–PerCPCy5.5, anti-CD4–BV510, and anti-CD8–BV570 for 15 minutes. Cells then were fixed with the FoxP3 staining kit (eBioscience) according to the manufacturer’s instructions, stained with anti-FoxP3–Pacific Blue, anti–IFN-γ–PECy7, anti-IL17–APC, anti-TNFα–BV650, and anti-IL22–PE for 30 minutes, washed in PermWash buffer (eBioscience), samples were acquired on an LSRII cytometer (BD, Franklin Lakes, NJ), and analyzed using FlowJo (Tree Star, Inc, Ashland, OR). Gating strategy for T and myeloid cells was performed as described previously.³²