Collagen based invasion assay kit

Invasion assay was performed 48 h after treatment with metformin using the collagen-based invasion assay kit from Millipore (Burlington, MA, USA) according to the manufacturer's protocol. Briefly, SF268 cells were starved in serum-free medium for 24 h before harvesting and resuspension in quenching medium (serum-free). Culture plate inserts were rehydrated using 300 μl of serum-free medium for 30 min at room temperature before plating the cells at a density of 0.6 × 10⁶ cells/ml. Specifically, 250 μl of the serum-free medium was removed from inserts and replaced by 250 μl of cell suspension. Inserts were then placed in a 24-well plate containing 500 μl of complete medium in each well before incubation for 24 h at 37°C in a CO₂ incubator. Following incubation, nonmigrating cells inside the upper cup were removed using a cotton swab and cells migrating through the membrane to the bottom surface of the cup were stained for 20 min at room temperature with 400 μl of cell stain provided with the kit. The stain was then extracted with extraction buffer and 100 μl of extracted stain was transferred to a 96-well plate suitable for colorimetric measurement using the Multiskan FC microplate ELISA reader, and optical density was measured at 560 nm.

Invasion assay was performed as previously described using the collagen-based invasion assay kit from Millipore (Burlington, MA) (Al-Koussa et al., 2020b (link); Al Haddad et al., 2020 (link); Al Hassan et al., 2018 (link)). Briefly, control and transfected ovarian cancer cells were starved for 24 h before resuspension in serum-free quenching medium and plating onto the hydrated inserts. The cells were then placed in wells containing complete medium (10% FBS) and incubated for 24 h. Following, the cells at the bottom surface of the inserts were stained with 400 μl of cell stain for 20 min at room temperature. After extracting the stain with the extraction buffer, 100 μl of the extracted stain were transferred to the wells of a 96-well plate. Finally, the optical density of each sample was measured at 560 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA).

Invasion assay was performed as previously described using the collagen-based invasion assay kit from Millipore (Burlington, MA) [9 ]. Briefly, control and transfected ovarian cancer cells were starved for 24 h before resuspension in serum-free quenching medium and plating onto the hydrated inserts. The cells were then placed in wells containing complete medium (10% FBS) and incubated for 24 h. Following, the cells at the bottom surface of the inserts were stained with 400 μl of cell stain for 20 min at room temperature. After extracting the stain with the extraction buffer, 100 μl of the extracted stain were transferred to the wells of a 96-well plate. Finally, the optical density of each sample was measured at 560 nm using the Varioskan microplate reader from ThermoFisher scientific (Massachusetts, USA).