Gentlemacs octo dissociator

Histones were extracted from cells or organs (disrupted using a gentleMACS^TM Octo Dissociator, Miltenyi Biotec; Bergisch Gladbach, Germany), run through SDS-PAGE and detected by Western blotting29 (link) using antibodies (diluted 1:1000) directed against acetylated histone 3 (H3) and H4 (06-599, 06-860) (EMD Millipore, Billerica, MA). Blots were revealed with the enhanced chemiluminescence Western blotting system (GE Healthcare, Little Chalfont, Great Britain). Images were recorded using a Fusion Fx system (Viber Lourmat, Collégien, France).

Colons were extracted from colitis-induced mice after euthanasia, and lamina propria lymphocytes (LPLs) were isolated using the Lamina Propria Dissociation Kit and gentleMACS^TM Octo Dissociator according to the manufacturer’s instructions (Miltenyi Biotec). For intracellular cytokine staining, the cells were cultured in the presence of brefeldin A solution, 50 ng/mL PMA and 1000 ng/mL ionomycin in RPMI 1640 medium containing 10% fetal calf serum, 10 mM HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μM 2-mercaptoethanol for 6 h at 37°C in 5% CO₂. After the incubation, intracellular cytokine staining was performed using anti-CD4, anti-IFN-γ and anti-IL-17 mAbs. Flow cytometric analysis was conducted using LSR with CellQuest software (Becton Dickinson).
To measure cytokine production by LPLs, the cells were stimulated with PMA and ionomycin in RPMI 1640 medium containing 10% fetal calf serum, 10 mM HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μM 2-mercaptoethanol for 6 h at 37°C in 5% CO₂. After the stimulation, levels of IFN-γ and IL-17 secreted into the culture medium were measured using cell-based assays with a BD FACSArray Bioanalyzer (Becton Dickinson).

Brain tumors were excised aseptically from mice, weighed, and mechanically diced. Single-cell suspensions were made using Brain Tumor Dissociation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany) and gentleMACS^TM Octo Dissociator (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The single-cell suspensions were filtered by a 70 μm filter and resuspended in FACS stain buffer. Red blood cells were lysed using an RBC removal solution. Cells were incubated with Fixable Viability Stain (BD Pharmingen, Franklin, NJ, USA). The cells were labeled with combinations of antibodies (Supplementary Table 3) against cell surface markers. At last, the cells were resuspended in FACS staining buffer and analyzed on the Beckman CytoFLEX (Brea, CA, USA). BD CompBeads were used to optimize fluorescence settings (BD Biosciences, Franklin, NJ, USA). Fluorescence-minus-one, unstained, and single-stained cells were also used to set gates.

The tissues were placed into gentleMACS tubes (Miltenyi Biotec, Bergisch Gladbach, Germen) filled with pure air (Taiyo Co. Ltd., Tokyo, Japan) immediately after sampling and then homogenized using the gentleMACS^TM Octo Dissociator (Miltenyi Biotec). Then, 2 ml of gas in the tube was collected through the rubber layer with a syringe. The hydrogen concentrations were subsequently measured using high-quality Sensor Gas Chromatography (SGHA-P1; FIS Co. Ltd., Hyogo, Japan). Each arterial blood sample was placed in a glass tube filled with the pure air, and 2 ml of gas was collected for measurement. The hydrogen concentration in each blood or tissue sample was defined as follows: Hydrogen concentration (ppb/g; V/V) = A/B × C; where A is the measurement value obtained using the Sensor Gas Chromatograph, B is the blood or tissue weight (g) and C is the volume of the gentleMACS tube (24 ml).

Astrocytes from MBH, cortex, and hippocampus were isolated using magnetic-activated cell sorting (MACS). Briefly, mice were euthanized by decapitation. Tissue blocks were swiftly collected, finely chopped into small pieces in ice-cold Dulbecco’s phosphate buffered saline, and then transferred to gentleMACS^TM C tubes. Subsequently, the tissues were dissociated into single-cell suspensions using the Adult Brain Dissociation kit (Miltenyi Biotec, 130107677) and the gentleMACS^TM Octo Dissociator (Miltenyi Biotec). After centrifugation, the cells were resuspended and labeled with an anti-ACSA-2 microbead kit (Miltenyi Biotec, 130097678) for astrocyte isolation. To validate the accurate isolation of region-specific astrocytes, we performed qPCR analysis on brain region-specific astrocyte markers such as Agt, Lhx2, and Emx2^{64 (link)} (Supplementary Fig. 9).