Transwell boyden chamber

The invasion ability of MCF-7 cells was tested in a Transwell Boyden Chamber (6.5-mm, Costar) as described previously (Wang et al., 2017 (link)). The polycarbonate membranes (8-μm pore size) on the bottom of the upper compartment of the Transwells were coated with 1% human fibronectin (R&D Systems 1918-FN, USA). The cells were harvested 12 h after transfection and suspended in FBS-free DMEM culture medium. Then, cells were added to the upper chamber (2 × 10⁴ cells/well). At the same time, 0.5 mL of DMEM containing 10% FBS was added to the lower compartment, and the Transwell-containing plates were incubated for 6 h in a 5% CO₂ atmosphere saturated with H₂O.

The migration ability of HUVEC was tested in a Transwell Boyden Chamber (6.5 mm, Costar). The polycarbonate membranes (8 μm pore size) on the bottom of the upper compartment of the Transwells were coated with 1% gelatin matrix. Cells were suspended in serum-free M-199 culture medium at a concentration of 4 × 10⁵ cells/ml, treated with or without simvastatin treated THP-1 MVs for 2 hr and then added to the upper chamber (4 × 104 cells/well). Simultaneously, 0.5 ml of M-199 with 10% FBS was added to the lower compartment, and the Transwell-containing plates were incubated for 4 hr in a 5% CO₂ atmosphere saturated with H₂O. At the end of the incubation, cells that had entered the lower surface of the filter membrane were fixed with 90% ethanol for 15 min at room temperature, washed three times with distilled water, and stained with 0.1% crystal violet in 0.1 M borate and 2% ethanol for 15 min at room temperature. Cells remaining on the upper surface of the filter membrane (nonmigrant) were scraped off gently with a cotton swab. Images of migrant cells were captured by a photomicroscope. Cell migration was quantified by blind counting of the migrated cells on the lower surface of the membrane, with five fields per chamber.

The capacity of HUVEC to migrate was determined using a Transwell Boyden chamber (Corning Costar, Cambridge, MA, USA) with 8.0 μm pore-size polycarbonate filters inserts in 24-well plates as previously described [18 (link), 19 (link)]. Cells (5× 10⁶/ mL, 100 μL) were seeded on the upper compartment of the chamber in the presence or absence of different concentrations of irisin with 5 μg/ml mitomycin C in serum-free M199. The lower compartment contained 600 μL of M199 supplemented with 10% FBS to stimulate cell migration. After incubation for 24 h at 37°C, the filter was removed and fixed by formaldehyde (3.7% in PBS) at room temperature for 20 min and then stained by 1% crystal violet at room temperature for 15 min, and the non-migrating cells were scraped off the upper chamber with cotton swabs. The membranes were washed twice in PBS and cells on the lower surface were counted and photographed with a fluorescent microscope (Olympus; Tokyo, Japan) in 5 random fields.