Polycarbonate membrane inserts with 8.0 μm pores

The effects of α-synuclein and CXCL12 on microglia migration were assessed using polycarbonate membrane inserts with 8.0 μm pores (Corning, USA) placed in a 24-well plate as previously described [25 (link)]. Images of the migrated cells on the lower surface of the insert were captured under a phase-contrast microscope. The number of transmigrated cells was determined using an inverted microscope at × 200 magnification with five fields of view using ImageJ software. The experiment was repeated three times.

Macrophage migration was assessed using a transwell assay in a 24‐well plate with polycarbonate membrane inserts with 8.0 μm pores (Corning). Briefly, 1 x 10⁵ of RAW264.7 cells were seeded in the insert (upper chamber) in 100 μL of medium with 0.1% BSA. Lower chambers were then filled with 600 μL of either conditioned medium, or medium with 0.1% BSA as a negative control. For some wells, antibodies against OPN (100 ng/mL) or CCL2 (100 ng/mL) were added into the conditioned medium. After culture for 24 h, the migrated cells on the lower surface of the membrane were stained with DAPI (Invitrogen) and imaged using an EVOS microscope (Thermo) and quantified using Fiji.
Macrophage polarization was assessed by culturing the RAW264.7 or BMDM in the conditioned medium for 24 h. Medium containing LPS and IFN‐gamma or IL‐4 and IL‐13 were used for M1/M2 control. After 24 h of treatment, cells were scraped off the dish and collected for RNA isolation or staining and Flow Cytometry analysis.