Far red plasma membrane fluorescent probe

Myelin debris was prepared as previously described [16 (link)]. Briefly, the mice were euthanized and the brains were cut into pieces (approximately 5 mm³) in 0.32 M sucrose solution on ice. The solutions were then homogenized and transfered to the top of the 0.83 M sucrose solution, taking care not to mix the two layers. After centrifugation at 100,000 rpm for 45 min at 4 °C, the crude myelin debris was collected from the interface of the two sucrose densities, dissolved in Tris·Cl buffer solution and homogenized for 30–60 s. After two rounds of centrifugation, the pellets were resuspended in sterile phosphate buffer solution (PBS) and centrifuged at 22,000 rpm for 10 min at 4 °C. Finally, myelin debris was resuspended in PBS to a final concentration of 100 mg/ml. Primary microglia pretreated with the CXCR2 inhibitor SB225002 (10 nM) and rCXCL5 (25 μg/ml) were stimulated with myelin debris (0.01 mg/ml) for different times. Myelin debris was stained with DiD (a far-red plasma membrane fluorescent probe, Beyotime, China) or DiI (a red plasma membrane fluorescent probe, Beyotime, China) for flow cytometry or immunofluorescence.

Cells were incubated with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine,4‐chlorobenzenesulfonate Salt (DiD, Far‐red Plasma Membrane Fluorescent Probe, Beyotime, C1039, China) for 20 min to label the cell membrane. Then, the cells were fixed with paraformaldehyde for 15 min and permeabilized with 0.2% Triton X‐100 for 10 min. Next, the cells were incubated with primary antibodies at 4 °C overnight. The following day, the cells were incubated with fluorescent secondary antibodies (Beyotime, China) for 1 h, and washed three times with PBS buffer. Then DAPI (Beyotime, China) was used to stain cell nuclei for 10 min. After washing with PBS buffer for three times, the samples were analyzed through confocal microscopy.