As before,
10 (link) 2 h before sacrifice, mice were intraperitoneally injected with methoxy‐X04 5 mg/kg (2 mg/ml dissolved 1 to 1 into dimethyl sulfoxide (DMSO) and resuspended 0.9% saline solution). After intracardial perfusion, microglia was isolated by mechanical dissociation and density gradient. The density gradient was generated resuspending the cell pellet in 70% Percoll in PBS overlayed with 37% Percoll solution in PBS and centrifuged at 2000 g for 20 min in a refrigerated centrifuge. Microglia was obtained by the 37‐70% Percoll gradient and washed 1:5 in ice‐cold PBS and spun at maximum speed in refrigerated microcentrifuge for 1 min. Microglia cells were isolated with FACSAria™ III cell sorter (BD Biosciences) after staining with CD11b‐BV650 (1:200 Biolegend, 141723), CD45‐BV786 (1:200, BD Biosciences 564225), and CX3CR1‐FITC (1:100, Biolegend, 149019). Microglia were gated as live (propidium iodide negative), CD11b+, CD45+, CX3CR1+ single cells, and Me‐X04+ and Me‐X04− microglial populations in transgenic 5xFAD mice were sorted separately for further analysis (Supplementary FigureS2 ). The flow cytometry results were analyzed using FlowJo™ Software v10.8 Software (Becton, Dickinson and Company; 2023).
10 (link) 2 h before sacrifice, mice were intraperitoneally injected with methoxy‐X04 5 mg/kg (2 mg/ml dissolved 1 to 1 into dimethyl sulfoxide (DMSO) and resuspended 0.9% saline solution). After intracardial perfusion, microglia was isolated by mechanical dissociation and density gradient. The density gradient was generated resuspending the cell pellet in 70% Percoll in PBS overlayed with 37% Percoll solution in PBS and centrifuged at 2000 g for 20 min in a refrigerated centrifuge. Microglia was obtained by the 37‐70% Percoll gradient and washed 1:5 in ice‐cold PBS and spun at maximum speed in refrigerated microcentrifuge for 1 min. Microglia cells were isolated with FACSAria™ III cell sorter (BD Biosciences) after staining with CD11b‐BV650 (1:200 Biolegend, 141723), CD45‐BV786 (1:200, BD Biosciences 564225), and CX3CR1‐FITC (1:100, Biolegend, 149019). Microglia were gated as live (propidium iodide negative), CD11b+, CD45+, CX3CR1+ single cells, and Me‐X04+ and Me‐X04− microglial populations in transgenic 5xFAD mice were sorted separately for further analysis (Supplementary Figure
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