Murine Splenocyte Phenotyping and Analysis

Spleens were removed from sacrificed mice according to Kalli et al.^{45 (link)}. Frequency of memory T lymphocytes was assessed ex vivo on splenocytes stained by the following conjugated mAbs: CD3-BV510 (BD PharMingen, Haryana, India), CD4-APC-eFluor 780, CD8-PE-Cy7, CD44- PE (Thermo Scientific,Waltham, MA, USA). To assess ex vivo the percentages of non-lymphoid APCs (monocytes and DCs), splenocytes were labelled with the following conjugated mAbs: CD80- FITC (Biolegend, San Diego, CA, USA), HLA-DR-PE, CD86-APC (Thermo Scientific,Waltham, MA, USA). The samples were analyzed by a FACS Canto II flow-cytometer (BD Bioscience, Frankin Lake, NJ, USA) using FACS DIVA software. In both analyses, splenocytes were stained with Live/dead fixable violet dead cell stain (Thermo Scientific,Waltham, MA, USA) to exclude dead cells. Statistical analyses were performed by the Mann-Whitney unpaired T test for nonparametric measures using the GraphPad Prism 4.0 Software (GraphPad Software, Inc, La Jolla, CA).