Lsr 3

Blood samples from each mouse were collected from the eyes by Sino-orbital puncture of mice using micro-capillary tubes. Blood samples were stored in clean and dry test tubes that contained ethylene diamine tetra acetic acid (EDTA). Red blood lysed with 1 ml of 1x BD FACS TM lysing solution (BD Bioscience, USA) for 10 minutes at room temperature and centrifuge at 500 x g for 5 minutes, discard the supernatant and re-suspend the pellet in 3 ml of PBS. We performed cell count and viability analysis with hemocytometer. Cells were transferred to FACS tubes (BD Bioscience, USA) and performed surface staining with 5 μl of mouse mAbs that are specific for mice epitopes: PerpCP anti-CD3 (BD Bioscience, USA) and FITC anti-CD56 (Abcam, UK) for 30 minutes in the dark at room temperature, following xed the cells with 100 μl of xation buffer and permeabilized with 100 μl of Intracellular staining perm buffer. We performed intracellular staining with 5 μl of APC anti-TNFα (BD Bioscience, USA) and PE anti-INF-g (BD Bioscience, USA) for 30 minutes in the dark at room temperature. Flow cytometry was executed on an LSR III (BD Biosciences, USA) using Diva Ó software.