Anti cd3e fitc

Mouse spinal cords were obtained at the peak of the 2nd attack, 5 days after onset of treatment. Spinal cords were mechanically disrupted in Hibernate A (Life Technologies, Grand Island, NY) using a stainless steel sieve, which was followed by enzymatic dissociation in 1 mL of Trypsin LE (Life Technologies) for 30 min at 37 °C. Cells were washed and resuspended in 10 mL of Percoll of ρ = 1.03 which was underlain in Percoll of ρ = 1.095 and centrifuged for 30 min at 500μg and 4 °C with slow acceleration and no brake. The top layer containing myelin was discarded, and leukocytes were collected from the interface between ρ = 1.03 and ρ = 1.095. After washing, cells were treated with Fc block (BD Bioscience, San Jose, CA), stained with the indicated anti-mouse antibodies [anti-CD45-v500 and 7-AAD (obtained from BD Bioscience), anti-CD3e-FITC, anti-CD4-PE, anti-CD19-efluor450 and anti-CD11b-APC (all obtained from eBioscience, San Diego, CA)] for 30 min at 4 °C, and analyzed on a BD FACS Canto II flow cytometer. Spinal cord-derived cells were stained and analyzed by pooling two to three mouse spinal cord cells per tube. Gating was determined by fluorescence-minus-one (FMO) with isotype-matched immunoglobulin control. Flow cytometry data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR).