Biocoat growth factor reduced matrigel invasion chamber

An invasion assay was performed using a BD BioCoat Growth Factor Reduced Matrigel Invasion Chamber (BD Biosciences, San Jose, CA, USA), according to the manufacturer's instructions. An equivalent number of cells (5×10⁴/well) was resuspended in serum-free medium, with or without CoCl₂ (200 µM), and plated in the upper insert. To further examine the effects of autophagy inhibition on invasion under hypoxia, another group of cells were incubated in the presence or absence of chloroquine (CQ; 10 and 20 µM; Sigma-Aldrich) with 200 µM CoCl₂. The insert was then placed inside a 24-well plate. The cells were incubated for 24 h at 37°C, following which the upper surface of the membrane in each insert was gently wiped with a cotton swab to remove all non-invading cells. The cells on the under surface were fixed with 4% paraformaldehyde for 15 min. The insert was subsequently placed in 0.1% crystal violet (Beijing Chemical Industry Group, Co., Ltd.) to stain the cells. The numbers of cells in seven randomly-selected fields of each filter were estimated by manual counting, and each experiment was performed independently at least three times. Leica-DM4000B microscope.

Stable DACT2-expressing or control HCT116 cells were transfected with 50 nM E-cadherin siRNA (sense: 5'-GAUUGCACCGGUCGACAAA dTdT-3') (RiboBio, Guangzhou, China) or control siRNA (RiboBio). Forty-eight hours post transfection, cells were collected for western blot and Transwell assays using a BD BioCoat Growth Factor Reduced MATRIGEL Invasion Chamber (BD Biosciences) as described previously.^{38 (link)}

A wound healing assay was employed as follows; a scratch was made on a uniform layer of cells using a sterile micropipette tip and cells were washed to remove debris. Photographs of the same area of the wound were taken after 4, 8, and 24 hours to measure the width of the wound. For the transwell migration assay, cells were resuspended in serum-free medium and seeded into the insert well of a 24-wells plate (8 μm pores, BD biosciences, Franklin Lakes, NJ) for 24 hours. The culture medium containing 10% FBS was used as a chemoattractant and placed in the bottom chamber. Cells were fixed in PFA (4%) and stained in crystal violet (0.5% in 20% methanol). Remaining cells in the upper chamber (non-migratory cells) were removed with a cotton swab. Adherent cells on the bottom of the membrane (migratory cells) were counted under a microscope (Olympus America Inc., Center Valley, PA) and Stereologer System (Dissector, Stereology Resource Center, Inc., Chester, MD). The invasion assay was performed under the same conditions using the BD BioCoat™ growth factor reduced MATRIGEL™ invasion chamber (BD biosciences).