GSM7060818: brain young Sham Day 2 replicate4 (male); Mus musculus; RNA-Seq
Brain single cell suspensions were obtained by enzymatic digestion with papain (Neural Tissue Dissociation Kit (P), #130-092-628, Miltenyi Biotec) following the manufacturer's instructions. Four to five hemispheres were pooled for each experiment. In order to increase cell viability and to preserve the transcriptional state during the generation of single-cell suspensions, Brilliant Blue G (BBG, P2X7 receptor antagonist, 1M, Sigma), actinomycin D (RNA polymerase inhibitor, 5g/ml, Sigma) and AP-5 (25 nM, NMDA receptor blocker, Tocris) were added to the dissociation solution. Next, cell suspension was filtered through a 70 m filter, resuspended in 30% Percoll (GE Healthcare)-HBSS containing 1M BBG, and spun for 10 minutes at 700 g. After gradient centrifugation, the myelin layer was removed, and the cell pellet was resuspended in 2%FBS-PBS buffer and filtered through a 40 m filter. Isolated cells were washed and resuspended in 100 l of blocking buffer per hemisphere to proceed for FACS staining and cell sorting. For isolation of peripheral leukocytes, mice were anesthetized with pentobarbital (100 mg/Kg, i.p.) and 0.5 ml of blood was drawn from the heart and transfer into tubes containing sodium heparin (20 Units). BBG (1M) and actinomycin D (5g/ml) inhibitors were added during the isolation procedure. For each experiment the blood from two mice (1 ml total blood) was pooled and erythrocytes were lysed. After erythrolysis, blood cells were resuspended in MACS buffer (PBS supplemented with 2% FBS, 2 mM EDTA; 300 μl/107 cells), incubated with a biotinylated Ter-119 antibody (Biolegend) and purified with anti-biotin microbeads according to the manufacturer's instructions (Miltenyi Biotec). Afterward, cells were washed and resuspended in 0.01% BSA-PBS at a concentration of 105 leukocytes /ml.