Uv blue live dead fixable stain

Animals were perfused with PBS and brains and spinal cords were removed. A single cell suspension was obtained and passed through a 70 μm strainer. Mononuclear cells were obtained by Percoll gradient (37%/70%) centrifugation and collected from the interphase. For intracellular cytokine analysis, cells were washed and stimulated with 5 ng/ml of PMA, 250 ng/ml of ionomycin the presence of brefeldin A (Golgi Plug reagent, BD Bioscience) for 4 hours. Cells were labeled with the UV-Blue Live/Dead fixable stain (Invitrogen) followed by surface staining (CD45, CD11b, CD4, CD8, TCRγδ, and TCRβ). Afterwards, cells were fixed, permeabilized and stained for intracellular IL-17A, IFNγ, and GM-CSF as described above. Alternatively, unstimulated CNS cells were surface labeled for CD45, CD11b, TCRβ, CD4, CD8, then fixed and stained for FoxP3, as above.