Multicolor Flow Cytometry for Mouse Splenocytes

For 10-color flow cytometric analysis, single cell suspensions from harvested spleens were prepared (as in Cheema et al13 (link)) and stained with fluorochrome-conjugated anti-mouse antibodies (PerCP-Cy-5.5 anti-mouse CD4, PE-Cy7 anti-mouse CD69, Alexa Fluor 647 anti-mouse FoxP3, Brilliant Violet 510 anti-mouse CD8a, Brilliant Violet 421 anti-mouse NK 1.1, Brilliant Violet 605 anti-mouse/human CD11b, APC-Cy7 anti-mouse CD11c, Alexa Fluor 700 anti-mouse Ly-6G/Ly-6C Gr-1, and FITC anti-mouse CD19), as well as appropriate isotype control antibodies, as described.14 36 (link) All antibodies were obtained from Biolegend. Zombie UV live/dead fixable viability kit was used to stain dead cells. We followed a ‘no-wash’ sequential staining protocol (Biolegend) to stain dead cells and for surface staining. Intracellular FoxP3 staining was performed following the FoxP3 intracellular staining protocol (Biolegend). Fluorescent minus one and single-color compensation controls were included for each color, as we described.14 36 (link) All samples were run in a LSRII flow cytometer (BD Biosciences) and flow cytometric data were analyzed by FlowJo software V.10.5.3 (Tree Star). Technician acquiring and gating the data were blinded to the treatments.