Biocoat matrigel invasion chamber

We performed cell transwell invasion and migration assays using 24‐well BD BioCoat Matrigel Invasion Chambers (8 μm pore size; BD Biosciences, SanJose, CA, USA). 1 × 10⁵ serum starvation OS cells were re‐suspended in 200 μL serum‐free medium and added to the upper wells of BD BioCoat Matrigel Invasion Chambers for invasion (with matrigel basement membrane matrix over the PET membrane) and migration (without matrigel basement membrane matrix). MEM (400 μL) containing 10% FBS was filled into the bottom chambers. After 24 hours of incubation, the cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 15 minutes. And then, the nonmigrated cells were wiped off from the surface of PET membrane with swab, the cells that migrate through the pores will be counted under the microscope and photographed in ten randomly selected fields. All the assays were performed in triplicate.

ARF6, NEDD9, and MT1-MMP knocked down HEC-1B and Ishikawa cells and their scrambled siRNA transfected cells were used to assess the effects of knockdown of these proteins on the invasive property of endometrial cancer cells. The Biocoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA) were rehydrated at 37°C for 2 h. Control and knockdown cells were detached by trypsin and resuspended in serum-free medium. Medium containing 10% fetal bovine serum was applied to the lower chambers of BD Biocoat Matrigel Invasion Chambers as chemoattractant and then cells were seeded on the upper chambers at a density of 2.5 × 10⁴ cells/well in 100 ml of serum-free medium. The chambers were incubated for 16–18 h at 37°C. At 18 h after plating, noninvading cells were removed from the upper surface of the membrane by scrubbing. The cells on the lower surface of the membrane were fixed for 2 min in 100% methanol and stained with 1% toluidine blue in 1% sodium borate for 2 min. Cells that invaded through the insert were counted in five random fields per slide. All slides were coded to avoid biased counting. The assay was run in triplicates.

Cell invasion was assessed using the Biocoat matrigel invasion chambers with 8 micron pores (BD, Biocoat matrigel invasion chambers, BD Biosciences, Franklin Lakes, NJ, USA). 2.5×10⁵ cells resuspended in OptiMEM without FBS were seeded into the matrigel coated 6-well inserts. OptiMEM plus 10% FBS was added to the lower chamber. After 48 hours, non invading cells located in the upper side of the chamber were wiped off using cotton swabs. Invaded cells were fixed in 100% methanol and stained with tolouidine blue. Invasion was measured by counting the number of cells in 10 microscopic fields (magnification 20X) and results are expressed as fold change of invasive cell subpopulation respect to proliferative one, which was set as 1 for definition.

BioCoatMatrigel Invasion Chamber (BD Biosciences-Discovery Labware, Inc Bedford, MA) was used to measure cell invasion according to manufacturer’s instruction. Briefly, A549 cells were stimulated with chronic CSE alone or in combination with GSK343 as described above. After 14 days of stimulation, cells were allowed to invade for 22 hours toward 5% FBS. The cells on the upper surface of the matrigel were removed, fixed with methanol and the membranes stained with Diff quick, and the cells adherent to the outer surface of membrane evaluated, counting at least six fields per filter in each group at 40X magnification.