Perivascular cells could induce microglial malfunction associated with Alzheimer's disease NatureNeuro
Microglia isolation for flow cytometry and FACS sort
PBS-perfused brains were quickly isolated from the skull and the hippocampus was dissected on ice using chilled instruments. For scRNA sequencing, brains were perfused with inhibitor cocktail including Actinomycin D and triptolide . Next, single-cell suspension was prepared using the Adult Brain Dissociation kit from Miltenyi Biotec , according manufacturer’s instructions and ref.
. Afterwards, cell suspension was filtered through a 70 μM cell strainer before mixing with debris removal solution. Cells were centrifuged , washed with ice-cold FACS buffer and incubated for 30 min at 4 °C with FACS buffer containing Fc block and primary antibody mix. Flow cytometry data were analyzed using FACSDiva software 4.0 and FlowJo 10 software .Spp1mice were decapitated, the brain was dissected from the skull and meninges were removed in ice-cold HBSS with 5% FBS.
Single-cell libraries were prepared using 10X Genomics Chromium Next GEM Single Cell 3′ kit v3.1, according to the manufacturer’s instructions. Cells were loaded on the chromium chip according to Supplementary Table; because sorted PVMs and fibroblasts were in low abundance, we loaded the whole sample, while for microglia cells we aimed for 5,000 cells per sample.
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