Multi-color FACS Analysis of Murine Immune Cells

Mouse colons and lymph nodes were processed for single cell suspension as previously published [44 (link)] and multi-color staining performed according to standard FACS staining protocols. Briefly, tissues were treated with collagenase (I, II, and IV, Sigma Aldrich, St. Louis, MO) and dispersed twice using the gentleMACs tissue dissociator (Miltenyi Biotech, Cologne, Germany). Cell suspensions were incubated in complete RPMI media for 24 hours before staining for flow cytometry. Multi-color staining was performed according to standard surface and intracellular FACS staining Biolegend protocols (Biolegend, San Diego, CA). Antibodies used in this study were anti-Ly6G-APC (clone1A8, Biolegend), anti-F4/80-PCP (PerCP/Cy5.5), (Biolegend, BM8), anti-IL-12p35-APC (eBioscience, 4010p35), anti-IL-10-FITC (Biolegend, JES5-16E3), anti-NK1.1-PCP (eBioscience, PK136), anti-CD3-FITC (Biolegend, 145-2C11), anti-CD4-PCP (Biolegend, GK1.5), anti-CD8-PCP (Biolegend, 53-6.7), anti-Tbet-FITC (Biolegend, 4B10), anti-IFNγ-PE (Biolegend, XMG1.2), anti-IL-17A-APC (Biolegend, TC11-18H10.1), and anti-IL-4-FITC (eBioscience, 11-7042-82). All samples were analyzed on a Guava easyCyte 8HT flow cytometer (EMD Millipore, Bellerica, MA, USA), and analyzed using FCS Express software (DeNovo Software, Los Angeles, CA, USA).