Gentlemacs dissociator with heater

After mice were transcardially perfused with ice-cold PBS, whole brains were isolated to obtain single cell suspensions using the Adults Brain Dissociation Kit, mouse and rat (Miltenyi Biotec, 130-107-677) according to the manufacturer’s protocol. Briefly, the brains were chopped into pieces in cold PBS, transferred into gentleMACS™ C Tubes (Miltenyi Biotec, 130-093-237) and digested using the provided enzymes with the gentleMACS™ Dissociator with Heaters (Miltenyi Biotec, 130-096-427). Tissue lysates were resuspended and filtered through 70μm cell strainers, which were subsequently rinsed with PBS. Samples were centrifuged at 1500 rpm for 10min and the supernatant was decanted. This was followed by gradient centrifugation with 30% Percoll (GE Healthcare Life Sciences, 17-0891-01) in PBS (v/v) (2750 rpm for 30 minutes, at 4°C, no brakes). Myelin was removed with a suction pump and the suspensions were filtered through 70 μm cell strainers and centrifuged at 1500 rpm, 4°C for 10 minutes. The samples were then ready for flow cytometry staining.

Subcutaneous tumours from mice were dissociated using a human tumour dissociation kit (Miltenyi Biotec, 130-095-929) with an enzyme cocktail and a gentleMACS dissociator with heaters (Miltenyi Biotec). The cell suspension was then strained through a 40 µm cell strainer (Corning, 352340). Samples were treated with 1× Red Blood Cell Lysis solution (Miltenyi Biotec, 130-094-183) for 10 min at 4 °C, and then spun down at 300g for 5 min and resuspended in 0.5% BSA in PBS. Last, mouse cells were removed from the sample using a Mouse Cell Depletion kit (Miltenyi Biotec, 130-104-694) following the manufacturer’s protocol.

At 7 weeks after glioblastoma transplantation, mouse brains were dissected, mechanically disaggregated with scissors, and dissociated using an Adult Brain/Lung Dissociation Kit (Miltenyi Biotech) and gentleMACS Dissociator with Heater (Miltenyi Biotech) in accordance with the established protocol. Dissociated cells were suspended in PBS containing 0.5% bovine serum albumin, and GFP⁺ glioma cells were analyzed/sorted using BD FACS Aria II (BD Biosciences) with BD FACSDiva Software, version 8.0.2 (BD Biosciences).