BMDMs were seeded in V-well plates and centrifuged at 600g for 6 min, and supernatant was removed. Cells were then incubated with Fc Block in FACS buffer for 15 min, followed by incubation with the primary antibody (ATP1A1) in 50-μl FACS buffer for 1 hour at 4°C. Cells were then fixed in 2% paraformaldehyde for 10 min at room temperature. Cells were then permeabilized and incubated for 15 min at room temperature. At this point, primary antibody was added for cytoplasmic staining for 1 hour at 4°C. Secondary antibody was added in 50 μl of FACS buffer for 1 hour at 4°C. Cells were then resuspended in FACS buffer and measured using BD Biosciences FACSCanto II.

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