Ow cytometry analysis

Brain tissue from HICT and SED healthy or pre-onset EAE mice were dissociated to single cell suspension, using the Neural Tissue Dissociation Kit (Miltenyi Biotec). Myelin was removed using Percoll (GE Healthcare) followed by microglia isolation using CD11b microbeads and MS columns (Miltenyi Biotec) according to manufacturer instructions. Degree of microglia enrichment was assessed by CD11b (BD Bioscience, M1/70) staining and ow cytometry analysis (Beckman Coulter). In all experiments at least 85% of isolated cells expressed CD11b.

An ADP/ATP Assay Kit (Sigma-Aldrich) was used in the ADP/ATP ratio assay. In brief, BV2 cells were seeded in a 12-well plate (1 × 10 5 cells/well). After LPS or/and compound stimulation for 16 hours, the cells were digested and collected in a centrifuge tube. Then, the cells were seeded in a 96-well plate with a white background (1 × 10 Coculture BV2 cells were cultured in a 6-well plate at a density of 2.0 × 10 5 cells/well. Twenty-four hours later, the supernatant was collected as conditioned medium prior to LPS stimulation, and the compounds were stimulated for 6 hours. MES23.5 dopaminergic neural cells were cultured in a 96-well plate (2.0 × 10 4 cells/well), and after 12 hours, the conditioned medium was added to MES23.5 for 24 hours. Then, cell viability was measured by MTT assay and ow cytometry.
Flow cytometry BV2 cells were treated with 2-DG for 30 min prior to lipopolysaccharide (LPS)-Alexa Fluor® 488 (L23351, Invitrogen, CA, USA) stimulation. After 2 hours, Cells were resuspended and washed in PBS for 3 times.
The cell-associated uorescence was measured by ow cytometry analysis (Beckman Coulter, Brea, CA, USA).