Transwell migration chambers

A scratch wound healing model was conducted to check the migratory ability of PC3 cells following the transfection treatment. PC3 cells were plated at a density of 1 × 105 cells in 2-well Lab-Tek Chamber Slide (Sigma-Aldrich). After overnight incubation, cells were transfected with mimics or miR-NC. Wounds were created in confluent cells using a 200 μL pipette tip. The cells were rinsed several times with media to remove any free-floating cells or debris and the speed of wound closure was monitored after 24 h by measuring the change in distance of the wound edges. Each experiment was conducted in triplicate and representative scrape lines were photographed using a phase contrast microscope (Leica). For the invasion assays, after 24 h transfection, 1 × 105 cells in serum-free media were seeded onto the transwell migration chambers (8 μm pore size, Millipore). The insert in the upper chamber was coated overnight with 1 mg·mL⁻¹ BD Matrigel Matrix (BD Biosciences, Milan, Italy). A medium containing 10% FBS was added to the lower chamber. After 24 h, the non-invading cells were removed with a cotton-tipped swab and cells at the bottom of the Matrigel were stained with May–Grunewald–Giemsa stain (Sigma-Aldrich). Stained cells were counted under a microscope (Leica) at 200X magnification in 5 random fields in each well. Experiments were independently repeated three times.

HGC-27 and MGC-803 cells were grown to confluence on 12-well plastic dishes and treated with miRNA mimics or Scramble. Then 24 hours after transfection, linear scratch wounds (in triplicate) were created on the confluent cell monolayers using a 200 μL pipette tip. To remove cells from the cell cycle prior to wounding, cells were maintained in serum-free medium. To visualize migrated cells and wound healing, images were taken at 0, 24, 48 hours. A total of ten areas were selected randomly from each well and the cells in three wells of each group were quantified.
For the invasion assays, after 24 hours transfection, 1 × 10⁵ HGC-27or MGC-803 cells in serum-free media were seeded onto the transwell migration chambers (8 μm pore size; Millipore, Switzerland) which coated with the upper chamber of an insert coated with Matrigel (Sigma-Aldrich, USA). Media containing 20 % FBS were added to the lower chamber. After 24 hours, the noninvading cells were removed with cotton wool, Invasive cells located on the lower surface of the chamber were stained with May-Grunwald-Giemsa stain (Sigma-Aldrich, USA) and counted using a microscope (Olympus, Tokyo, Japan). Experiments were independently repeated three times.

Cells were placed in the top chamber of transwell migration chambers (8 μm; Millipore, Billerica, MA, USA). After 48 h, cells which had not migrated to the lower chamber were removed from the upper surface of the transwell membrane with a cotton swab. Migrating cells on the lower membrane surface were fixed, stained, photographed and counted using a microscope at ×100. Experiments were assayed in triplicate, and ≥5 fields were counted in each experiment.

MG-63 cells were grown to confluence in 12-well plastic dishes and were treated with miRNA mimics, inhibitors or Scrambled. Then, 24 hours after transfection, linear scratch wounds (in triplicate) were created on the confluent cell monolayers using a 200 µL pipette tip. To remove cells from the cell cycle prior to wounding, cells were maintained in serum-free media. To visualize migrated cells and wound healing, images were taken at 0, 24 and 48 h. A total of ten areas were selected randomly from each well, and the cells in three wells from each group were quantified.
For these invasion assays, 24 hours after transfection, 1×10⁵ cells in serum-free media were seeded in transwell migration chambers (8 µm pore size; Millipore, Zürich, Switzerland). The upper chamber of thesetranswell inserts was coated with Matrigel (Sigma-Aldrich, St. Louis, MO, USA). Medium containing 20% FBS was added to the lower chamber. After 24 hours, the non-invading cells were removed with cotton wool. Invasive cells located on the lower surface of the chamber were stained with May-Grunwald-Giemsa stain (Sigma-Aldrich, St. Louis, MO, USA) and then were counted using a microscope (Olympus, Tokyo, Japan).

To estimate the competence of cell migration, cell transwell assays were designed, and executed principally with the application of the Transwell migration chambers (8 μm pore size, Millipore Corporation, Billerica, MA). Firstly, cells were cultured on 6-well plates and transfected with miR-770-5p inhibitor/mimics, SRGAP1 siRNAs, MEG3 siRNAs, MEG3-overexpression lentivirus and negative control. Cells were suspended with serum-free medium, then in the upper chamber 100μl cell suspension was seeded, and in the lower chamber 600ul medium with 10% FBS. After incubated for 24 to 48 hours, cells were dyed with crystal violet staining solution (Beyotime, Nantong, China). We used Image-pro Plus 6.0 to photograph under 40×magnification (five views per well) and counted migrated cells while cell numbers of normal control were normalized to 1. Besides, CCK8 assay (Beyotime, Nantong, China) was applied to test the cell proliferation. The TECAN infinite M200 Multimode microplate reader (Tecan, Mechelen, Belgium) was used to measure the absorbance at 450 nm. All experiments were repeated three times independently.

Cell transwell assay, which was designed to evaluate the capacity of cell migration, was mainly performed with the application of the Transwell migration chambers (8 μm pore size, Millipore Corporation, Billerica, MA). Firstly, two cell lines were cultured in six-well plates and transfected with miR-206 inhibitor/mimics or SDPR siRNA. After 48 h of transfection, cells were harvested with serum-free medium as single-cell suspension and 100 μl of cell suspension was seeded in the upper chamber (1 × 10⁶ cells/ml), along with the lower chamber filled with the 600 μl DMEM medium with 10% FBS. After incubation from 24 h to 48 h, the cells were stained with crystal violet staining solution (Beyotime, Nantong, China) after cell fixation in 95% methyl aldehyde. The images of migrated cells were captured and counted by Image-pro Plus 6.0 with the amount of normal control cells standardized to 1. Accompanied with the transwell assay, CCK8 assay was conducted to confirm the cell proliferation after 24 h transfection. Transfected cells were planted into 96-well plates and add CCK8 reagent into each well for 1 h incubation at 37°C, which was evaluated by 450 nm absorption measured by TECAN infinite M200 multimodemicroplate reader (Tecan, Mechelen, Belgium). Experiments of cell transwell and proliferation were performed in triplicate independently.