Matrigel coated membrane matrix

A wound-healing assay was performed to assess the cell migration ability. An artificial wound was created on a confluent cell monolayer 24 hours after transfection. Images were taken at 0, 24, 48 hours and percentage of open wound was calculated.
HGC-27 and MGC-803 cells were seeded onto a Matrigel-coated membrane matrix (BD Bioscience) present in the insert of a 24 well culture plate. FBS was added to the lower chamber as a chemoattractant. After 24 hours, invasive cells located on the lower surface of chamber were stained with the 0.1% crystal violet (Sigma) and counted.

Wound-healing assays were performed to assess cell migration. An artificial wound was created 24 hours after transfection by using a 200-µL pipette tip on the confluent cell monolayer. Mitomycin C was then added to the culture wells. To visualize cell migration and wound healing, images were taken at 0, 24, and 48 hours.
Invasion assays were performed by examining the ability of cells to pass through a Matrigel-coated membrane matrix (BD Biosciences). Cells were seeded 24 hours after transfection onto a Matrigel-coated membrane matrix that was present in the insert of a 24-well culture plate. Fetal bovine serum was added to the non-invading cells that were removed. Invasive cells that were located on the lower surface of the chamber were stained with 0.1% crystal violet (Sigma) and were then counted.

SGC-7901 and HGC-27 cells were grown to 50–70% confluence and transfected with miR-NC, miR-22 mimics, si_con, si_MMP14#1, si_MMP14#2, si_Snail#1, si_Snail#2, pcDNA3.1 vector, pcDNA3.1-MMP14 vector or pcDNA3.1-Snail vector, co-transfected with miR-22 mimics and pcDNA3.1-MMP14, or co-transfected with miR-22 mimics and pcDNA3.1-Snail, respectively. AGS cells were transfected with miR-NC or anti-miR-22 inhibitor. Twenty-four hours posttransfection for invasion assay, cells were seeded onto a Matrigel-coated membrane matrix (BD Biosciences, San Jose, CA, USA) present in the insert of a 24-well culture plate (Costar, Corning, NY, USA). In the lower chamber, 500 μl DMEM with 10% fetal bovine serum was added as chemoattractant. After 24 h, the noninvading cells were gently removed with a cotton swab. Invasive cells located on the lower surface of chamber were stained with the 0.1% crystal violet and counted under a microscope in five predetermined fields (× 200). The procedure for the cell migration assay was similar to the cell invasion assay, except that the transwell membranes were not precoated with matrigel. Cells adhering to the lower surface were counted the same way as the cell invasion assay. All assays were independently repeated at least three times.

A 1.5-mL base layer of agar (0.5% agar in DMEM with 10% FBS) was allowed to solidify in a six-well flat-bottom plate before the addition of a 1.5 mL cell suspension containing 4,000 cells in 0.35% agar in DMEM with 10% FBS. The cell-containing layer was then solidified at 4°C for 20 minutes. Colonies were allowed to grow for 21 days at 37°C with 5% CO₂ before imaging.
A wound-healing assay was performed to assess effects on cell migration. An artificial wound was created on a confluent cell monolayer using a 200μL pipette tip 24 hours after transient transfection. Mitomycin C was added to the culture wells (final concentration for PANC-1, 10 μg/mL; for MIA PaCa-2, 20 μg/mL) to prevent proliferation. Images of the migration/wound healing process were captured at 0, 12, 24, 36, 48, and 60 hours [28 (link)].
Invasion was evaluated by the ability of cells to pass through a Matrigel-coated membrane matrix (BD Biosciences, NJ, USA). Twenty-four hours after transient transfection, cells were seeded onto a Matrigel-coated membrane matrix in a 24-well culture plate. Fetal bovine serum was added to the lower chamber as a chemoattractant. After 24 hours, non-invading cells were removed. Invasive cells, located on the lower surface of the chamber, were stained with 0.1% crystal violet (Sigma—Aldrich, MO, USA) and counted.

A wound-healing assay was done to assess cell migration. An artificial wound was created 24 hours after transfection using a 200-µL pipette tip on the confluent cell monolayer and mitomyclin C was added to the culture wells. To visualize migrated cells and wound healing, images were taken at 0, 24 and 48 hours.
Invasion assay was evaluated by the ability of cells passing through Matrigel-coated membrane matrix (BD Biosciences). Cells were seeds onto a Matrigel-coated membrane matrix present in the insert of a 24-well culture plate 24 hours after transfection. Fetal bovine serum was added to the noninvading cells were removed. Invasive cells located on the lower surface of the chamber were stained with 0.1% crystal violet (Sigma) and counted.