Spleen and lymph nodes (cervical and mesenteric) from naïve DA rats were harvested and single cell suspension prepared as described (36 (link), 37 (link)). RBCs were lysed from single cell suspensions from spleen using lysis buffer (Biolegend). Cell suspensions from spleen and lymph nodes were combined and cells were resuspended in PBS/0.4% BSA (MultiGel, Biosciences, Castle Hill, NSW, Australia). Cells were subjected to immunostaining using mAb against lymphocyte markers and subjected to enrichment of lymphocyte subsets.
Flow cytometry data was acquired on a FACScan or FACS Canto II (Becton Dickinson, San Jose, CA) using CellQuest or FACS DIVA (BD), as described (28 (link), 35 (link)). In some experiments doublets were excluded before cells in lymphocyte gate were subjected to dead cell exclusion and live cells were analyzed for expression of cell surface markers. Foxp3+ cells analysis did not include a live cell gate, as the dead cell marker and Foxp3 were both on the APC channel.
Flow cytometry data was acquired on a FACScan or FACS Canto II (Becton Dickinson, San Jose, CA) using CellQuest or FACS DIVA (BD), as described (28 (link), 35 (link)). In some experiments doublets were excluded before cells in lymphocyte gate were subjected to dead cell exclusion and live cells were analyzed for expression of cell surface markers. Foxp3+ cells analysis did not include a live cell gate, as the dead cell marker and Foxp3 were both on the APC channel.
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