Gentlemacs octo dissociator with heater

Mice from postnatal day 1 (P1) to P3 were sacrificed by decapitation. The brain was dissected and stored in DMEM before it was transferred to C-Tubes (Miltenyi Biotec). For dissociation, the multi-tissue dissociation kit (Miltenyi Biotec) and the gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec) with the program NKDT1 were used per the manufacturers’ protocols. Cell suspensions were filtered through a 70 µm filter (Sarstedt, Nümbrecht, Germany) and diluted in PBS containing 0.5% BSA. For the isolation of OPCs, the CD140a (platelet-derived growth factor receptor (PDGFR)α) microbead kit was used (Miltenyi Biotec) in combination with the QuadroMACS™ Separator (Miltenyi Biotec) with LS Columns (Miltenyi Biotec). In brief, columns were activated with 0.5% BSA/PBS solution. After samples had been applied, columns were washed and eluted with the same solution. OPCs were cultured in DMEM/F12 (Thermo Fisher Scientific) containing 1% N2 supplement, 2% B27 supplement (Thermo Fisher Scientific), 1% penicillin/streptomycin, 10 ng/mL PDGF-AA (Miltenyi Biotec), and 10 ng/mL FGF-2 (Miltenyi Biotec). Cell culture flasks were coated with 0.0075 µg/mL poly-D-lysine (Merck), and 24-well plates were coated with 0.0001 PORN before the cells were seeded.

Transcription and translation inhibitors were included during cell preparation, as previously described (Marsh et al., 2020 ) with some minor modifications. Actinomycin D (#A1410, MilliporeSigma) was reconstituted in DMSO to a concentration of 5 mg/ml before being aliquoted and stored at −20°C protected from light. Triptolide (#T3652, MilliporeSigma) was reconstituted in DMSO to a concentration of 10 mm before being aliquoted and stored at −20°C protected from light. Anisomycin (#A9789, MilliporeSigma) was reconstituted in DMSO to a concentration of 10 mg/ml before being aliquoted and stored at 4°C protected from light. For the samples to be treated with inhibitors, 2 μl each of Actinomycin D, Triptolide, and Anisomycin stocks were added to the initial Enzyme Mix 1 before dissociation for a final concentration of 5 μg/ml, 10 μm, and 10 μg/ml, respectively. The transcription and translation inhibitors were only included during the enzymatic and mechanical dissociation step for 30 min at 37°C on the gentleMACS Octo Dissociator with Heaters (#130-096-427, Miltenyi Biotec) using the 37C_ABDK_01 program. The transcription and translation inhibitors were not present in the dissection or sorting buffers, as the cell preparation was kept at 4°C for all other steps.

Freshly harvested tumors from Trem1^+/+ or Trem1^–/– mice were processed into a single-cell suspension using gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech) in combination with the tumor dissociation kit (Miltenyi Biotech). Cells were stained with fluorochrome–conjugated antibodies according to the manufacturer’s instructions. For surface staining, cells were prepared and suspended in PBS and incubated with following antibodies (all from BioLegend) at 4°C for 45 minutes in dark: TruStain FcX (clone: 93, 101319, 1:50 dilution), anti-F4/80-APC (clone: BM8, 123116, 1:100 dilution), anti-F4/80-FITC (clone: BM8, 123108, 1:100 dilution), anti-CD11b-APC (clone: M1/70, 101212, 1:100 dilution), anti-CD11b-PE (clone: M1/70, 101208, 1:200 dilution), anti-CD11b-APC/Cy7 (clone: M1/70, 101226, 1:100 dilution), anti-Gr1-APC/Cy7 (clone: RB6-8C5, 108423, 1:100 dilution), anti-Ly6C-PE (clone HK1.4, 128007, 1:200 dilution), anti-Ly6C-APC/Cy7 (clone: HK1.4, 128026, 1:100 dilution), anti-Ly6G-PE (clone: 1A8, 127608, 1:200 dilution), and anti-Ly6G-APC/Cy7 (clone: 1A8, 127624, 1:100 dilution). Cells were acquired on the Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and data were analyzed on FlowJo v10.0.

Following perfusion with ice-cold 1× DPBS containing 5 μg/ml actinomycin D, half brains were dissected, and the cerebellum was removed. Tissue was briefly stored on ice in RPMI 1640 containing 5 μg/mL actinomycin D, 10 μM triptolide, and 27.1 μg/mL anisomycin until subsequent perfusions were completed. Tissue dissociation was then performed utilizing the Tumor Dissociation kit, human (Miltenyi) and the gentleMACS Octo Dissociator with Heaters (Miltenyi) according to manufacturer guidelines with modifications. Briefly, tissue was cut into ~1 mm³ pieces and placed into the C-tubes with the kit’s enzymes, 5 μg/mL actinomycin D, 10 μM triptolide, and 27.1 μg/mL anisomycin and samples were dissociated using the preprogrammed protocol. Following enzymatic digestion, samples were strained through a 70 μm filter and pelleted by centrifugation. Myelin and debris were removed by resuspending the pellet in 8 mL 23% Percoll, overlaid with 2 mL of 1× DPBS, spinning at 400 × g for 25 min at 4 °C, with acceleration and brake set to 0, and discarding the myelin band and supernatant.