Dsc hx90

To determine the role of MTHFD2 in breast cancer malignancy, in vitro functional assays were performed to compare the effect of MTHFD2 overexpression and MTHFD2 deficiency in MCF-7 cells.
Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8; Abmole Bioscience, Inc.). Briefly, cells were seeded into 96-well plates at the density of 1,000 cells per well for 1, 3 and 5 days. Cells were incubated with CCK-8 and incubated at 37˚C for 2 h. Absorbance was read at 450 nm using a microplate reader.
For the colony formation assay, 2x10² cells were seeded into six-well plates for 14 days at 37˚C and 5% CO₂. Methanol and 0.1% crystal violet were used for fixation and staining of colonies at room temperature, respectively. The number of colonies was imaged using a camera [DSC-HX90; SONY (China), Co. Ltd.] and quantified.

Briefly, the cells were digested, plated into 6-well plates at a density of 500 cells/well and cultured with DMEM supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37°C for 2–3 weeks. The culture medium was replaced every 2 days. The cells were then immobilized with 4% paraformaldehyde for 15 min, and soaked in Giemsa stain (G4640, Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature and the cells were then washed twice with ultra-pure water. Images were acquired using a camera [DSC-HX90, SONY (China), Co. Ltd.].

Single cells at low density (52.63 cells per cm²) were seeded over a coating of 0.6% agarose solution containing supplemented media in normal conditions. The cells were allowed to sit for 10 min at room temperature and were then covered with a 0.3% agarose solution. Plates were incubated at 37°C in a 5% CO₂ humidified atmosphere and media were replaced every 3-4 days. After 15 days, The cells were then immobilized with 4% paraformaldehyde for 15 min, and soaked in Giemsa stain (G4640, Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature and the cells were then washed twice with ultra-pure water. Images were acquired using a camera [DSC-HX90; SONY (China), Co., Ltd.].