Transwell insert chamber

Transwell insert chambers (Corning Incorporated) coated with Matrigel (Solarbio Beijing, China) was employed for the analysis of cell invasion capacity. Briefly, A2058 and A375 cells (5 × 10⁵ cells/well) were digested in DMEM (Gibco) and seeded in the top chamber. DMEM (Gibco) including 10% FBS (Gibco) was added to the bottom chamber. Twenty-four hours later, cells that are still on the upper chamber were removed and cells that invaded to the lower chamber were fixed with paraformaldehyde (Sangon), stained with crystal violet (Solarbio), and then estimated under a microscope (Olympus, Tokyo, Japan).

Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8, Promotor, China) according to the manufacturer's instructions. CCK-8 reagent was added in the 96-well plates at 37°C for 2 hours to detect the optical density (OD) value at 450 nm. The migration and invasion assays were performed as described before by using transwell insert chambers (8 mm pore size, Corning, USA) [40 (link)].

Breast cancer cell invasive ability was measured by transwell insert chambers (Corning, NY, USA) pre-coated with Matrigel (Corning Inc.). In brief, 2 * 10⁴ cells were seeded into the upper chamber containing 200 µl serum-free medium, and 500 μl culture medium with 20% FBS was added into the bottom chamber. After 24 h, invading cells were fixed with 4% paraformal-dehyde and stained with 0.1% crystal violet. Cells were counted using a microscope for 5 fields randomly at 100× magnification.

Cell proliferation was assayed using Cell Counting Kit-8 (Dojindo, Japan) according to the manufacturer's instructions. Cells were seeded at a density of 2000 cells per well in 96 well plates. After AGAP2-AS1 siRNAs and negative control (NC) transfection for 24 h, cells were evaluated by measuring the absorbance at 450 nm. Cells were transfected with siRNAs targeting AGAP2-AS1 for 48 h and then about 1000 cells were plated in each well of the 12-well plate and maintained for 2 weeks to form colony.
Cell migration assay were performed by using Transwell insert chambers (8μm pore size, Corning, USA). About 2 × 10⁴ cells were seeded into the upper chamber in serum free medium in triplicate. The lower chamber was filled with 600μl medium containing 10% fetal bovine serum (FBS). After incubation for 4 h, cells migrating to the lower surface of membrane were fixed using paraformalclehyde and stained with 0.1% crystal violet. For invasion assay, Matrigel Invasion Chambers in the 24-well plates were used.