Pbs 1x

Following seven days upon initiation of the treatment, mice were weighed and deeply anesthetized with a ketamine/xylazine cocktail accordingly. Immune cells were isolated from whole brain homogenates as follows. Briefly, mice were transcardially perfused with ice-cold PBS 1x (Gibco), and brains were collected in DMEM (Gibco) supplemented with sodium pyruvate (Gibco) and a penicillin, streptomycin and glutamine cocktail (Gibco), gently mechanically disaggregated and resuspended in PBS 1x containing 3 mg/ml of collagenase D (Roche Diagnostics) plus 10 μg/ml of DNAse (Sigma-Aldrich) for an enzymatic homogenization. After a 30 min incubation, brain homogenates were filtered with 40 μm pore size cell strainers (BD Biosciences), centrifuged 8 min at 1800 r.p.m., washed with PBS 1x, and resuspended in 6 ml of 38% isotonic Percoll ® (GE Healthcare) before a 25 min centrifugation at 800 G without neither acceleration nor brake. Myelin and debris were discarded. Cell pellets containing total brain immune cells were collected, washed with DMEM supplemented with 10% fetal bovine serum (Gibco), and cell viability was determined by trypan blue exclusion using a Neubauer's chamber. Finally, cells were labeled for subsequent flow cytometric analysis.