Multiparameter Flow Cytometry Analysis

Cells were initially blocked with anti-mouse CD16/32 (eBioscience) then stained with fluorochrome-conjugated antibodies following our previous procedures (30 (link)). Briefly, after blocking of Fc receptors, cells were stained with fluorochrome-conjugated antibodies on ice in the dark for 15 min. Exclusion of dead cells was achieved with Zombie Aqua (BioLegend) staining. For quantification of T cells in total splenocytes, the following anti-mouse antibodies were used: CD3-APC, CD4-PE-Cy7, CD8-PerCP-Cy5.5, CD44-PE, CD62L-APC-Cy7, and CD69-Pacific blue (BioLegend). The same panel of antibodies were used for in-vitro activated splenocytes. For analysis of apoptosis, FITC-Annexin V Apoptosis Detection Kit (BioLegend) was used together with propidium iodide according to the manufacturer’s procedures. Splenic Gr-1+ cells in the CD11b+CD11c− myeloid population were analyzed using the following anti-mouse antibodies: CD11c-APC, CD11b-PerCP-Cy5, PD-L1-PE-Cy7 (BioLegend), and Gr-1-V540 (BD Bioscience). Analysis was performed with a BD FACSAria II flow cytometer (BD Biosciences). Flow cytometry data were analyzed with FlowJo.