Fitc conjugated anti mouse il 17a

Preparation of individual cell suspensions from EAE mouse spleen for flow cytometric analysis was performed as described before.^{47,48 (link)} Mice were killed and dealt with 75% alcohol pre-prepared, and then spleens were taken out and cut into fractionlets. Spleen tissue was crushed through the cell strainer in 2 mL HBSS and suspended cells were collected by centrifugation at 1500 rpm for 10 min. Ammonium–Chloride–Potassium (ACK) lysing buffer was used for the lysing of red blood cells in the mouse lymphocyte preps. Cells were washed with 3 mL HBSS three times, and then resuspended in RPMI-1640 with 10% FBS. For intracellular cytokine staining, the cells were stained with APC anti-mouse CD4 (eBioscience, CA, USA) at 4 °C for 1.5 h, and then fixed and permeabilized with 4% paraformaldehyde and 0.5% Triton-X100. Thereafter, the cells were stained with fluorophore-conjugated monoclonal antibody FITC-conjugated anti-mouse IL-17A (eBioscience, CA, USA) at 4 °C for 2.5 h. Flow cytometry was performed on a BD LSRII (BD Biosciences) instrument and analyzed using FlowJo (Tree Star, Inc., Ashland, OR).