Z2 coulter cell

Mononuclear cells from lymphoid tissue and the CNS were prepared as described previously [29 (link)], and all cell counts were performed using a Z2 Coulter cell and particle counter (Beckman Coulter, Miami, FL, USA). For T helper cell phenotyping, cells were resuspended at 1–5 × 10⁶ cells/ml in complete RPMI medium containing 50 ng/ml PMA and 1 μg/ml ionomycin. Four microliters of Golgistop (BD Bioscience) was also added for every 6-ml cell culture medium. Cells were seeded in 24-well plates at 5 × 10⁶ cells per well and incubated for 5 h at 37 °C with 5% CO₂. Cells were then harvested and counted, and intracellular cytokine staining performed on 3 × 10⁶ cells using PE-Cy7 anti-rat CD4, FITC anti-rat IFN- γ, PE anti-rat IL-4, and APC anti-mouse/rat IL-17A (eBioscience); antibodies were purchased from BD Biosciences except where stated. For analysis of regulatory T cells, 3 × 10⁶ splenocytes were stained with APC anti-rat CD4 and PE anti-rat CD25 (BD Bioscience), followed by intracellular staining with PE-Cy7 anti-mouse/rat Foxp3 (eBioscience). Following staining, cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences) and data analyzed using Flowlogic Software (Inivai Technologies, Mentone, Australia).

Mononuclear cells from lymphoid tissue and the CNS were prepared as described previously [30 (link)], and all cell counts were performed using a Z2 Coulter cell and particle counter (Beckman Coulter, Miami, FL, USA). For naïve T cell staining, cells were stained with appropriately diluted cell surface antibodies purchased from BD Bioscience. Analysis of the regulatory T cells (Treg) was performed with 3 × 10⁶ cells using a FoxP3 staining set (eBioscience) according to the manufacturer’s instructions. For T helper cell phenotyping, cells were resuspended at 5 × 10⁶ cells/ml in complete RPMI medium containing 50 ng/ml PMA and 1 μg/ml ionomycin. Four microliters Golgistop (BD Bioscience) was also added for every 6 ml cell culture medium. Cells were seeded in 24-well plates at 5 × 10⁶ cells per well and incubated for 5 h at 37 °C with 5 % CO₂. Cells were then harvested and counted, and intracellular cytokine staining was performed with 3 × 10⁶ cells using a mouse Th1/Th2/Th17 phenotyping kit (BD Bioscience), as per the manufacturer’s instructions. Cells were then analyzed using a FACSCanto II flow cytometer.