Transwell chambers 3 μm pore

The migration capacity of macrophages was measured in transwell chambers (3 μm pore, Corning). HDPC monolayers were incubated with serum-free DMEM with or without EZH2 (20 ng/ml), CCL2 (10 μg/L), EZH2+CCL2, and EZH2+anti-CCL2 (100 μg/L) treatment for 48 hours in a 5% CO₂ incubator at 37°C before collection of supernatants. After overnight culture in serum-free RPMI1640 medium, 200 μl of macrophages was resuspended (2 × 10⁶/ml) and added to the upper chamber in serum-free RPMI 1640 medium, and 600 μl of the supernatant of untreated or treated HDPCs was placed into the bottom wells. After 4 hours of incubation, nonmigrating cells on the upper surface of the membrane were removed, and the cells that migrated to the underside of the polycarbonate membrane were fixed with ethanol and stained with 1% crystal violet for 30 min. The mean of triplicate assays for each experimental condition was used for analysis.

For the 2D assay, cell culture inserts and the Chemotaxis and Migration Tool 2.0 (Ibidi, Madison, WI) were used. Inserts were coated with 20 μg/ml laminin (R&D Systems). PKH26-red-fluorescent-ECs in EGM media were plated on one side of the insert, and GFP-CSCs in complete NBM on the other side. Subsequently, the media was replaced with complete NBM, the insert removed, and live-imaging of cells into the gap initiated using a 10X objective lens and a motorized-stage (Leica-DMI6000-ImageEM/Orca-R2-ImageProPlus). Manual Tracking (Image J) was used to trace EC paths into the 500 μm gap [60 (link)]. ECs trajectories were projected onto the XY plane and the average velocity calculated for individual and total trajectories [60 (link)].
For analysis of CSCs/ECs migrating into the wound gap, an automated algorithm was generated by ImageIQ (Cleveland, OH) designed to batch process and analyze time-lapse, multi-channel stacks within ImagePro-Plus-7.0.
For the Transwell^® assay, cells were seeded on the upper filter surface (Transwell chambers, 3-μm pore, Corning), allowed to migrate, cells on the upper surface removed, and cells on the lower filter surface washed, fixed [11 (link)], photographed and counted in 5-10 fields with a 20X objective (Leica-DFC425C-QImaging-Q15729-QCapturePro).