8 mm pore transwell inserts

To assay the migration and invasion of cells, 5×10⁴ cells were suspended in 150 ml of serum-free medium and seeded onto 8-mm Pore Transwell Inserts (Corning, Corning, NY) for the migration assay or 8-mm Pore Transwell Inserts coated with Matrigel for the invasion assay. The lower chambers were filled with 800 ml of complete medium. To terminate the migration of the cells, the cells on the Transwell Inserts were fixed with 4% paraformaldehyde/PBS for 30 min. Subsequently, the fixed cells were stained with hematoxylin solution (Sigma-Aldrich, St. Louis, MO) for 1 hr. After wiping off the cells on the upper side of the filter on the Transwell Inserts using cotton swabs, microphotograms of the cells migrated on the lower side of the filter were taken using light microscopy. Cells migrated or invaded the lower side of the filter were manually counted from the microphotograms. The mean cell number was obtained from five randomly selected squares of same area per the transwell insert and were compared statistically.

4.0×10³ cells were seeded into 96-well-plate in quintuplicate. After 72 hours of incubation, the absorbance was measured at 450 nm by adding a CCK8 reagent (Boster Biological Technology) to detect cell proliferation. Cell migration was evaluated by scratch assays as described previously [25 (link)]. After 16 hours of incubation, the migrated area was photographed and calculated by ImageJ software. For invasion assay, 8 mm pore transwell inserts (Corning Life Sciences) were coated with 75μl Matrigel matrix (Corning, 200 μg/mL) as upper chambers. 700μl DMEM medium with 20% FBS was added into the 24-well-plate. Meanwhile, 5.0×104 cells were suspended in a serum-free medium and were added to the inserts which were placed in a 24-well-plate. After 48h, the cells were fixed with 10% formalin and stained with 0.1% crystal violet solution. The numbers of invaded cells were counted at 200x magnification.

AML cell lines KG-1a was ordered from the Shanghai cell bank of the Chinese Academy of Sciences and cultured in RPMI 1640 (Gibco) supplemented with 20% fetal bovine serum. Cells were maintained at 37 °C in a 5% CO₂ incubator. KG-1a was precultured with 10 ng/ml TGFβ1 (PeproTech) for 48 h and used for Cell Counting Kit-8 (CCK8) assay (Beyotime Institute of Biotechnology, China), apoptosis detection (Apoptosis Detection Kit, BestBio), cell cycle detection (COULTER DNA PREP Reagents Kit, Beckman). Cell cycle phase distributions were generated by fitting DNA content histograms to applicable models using ModFit LT software for Windows (Version 5.0). KG-1a cells were stained with Senescence‐associated‐β‐Galactosidase (SA‐β‐Gal) staining (Beyotime) according to the instructions after TGFβ1 treatment for 7 days, and cells that stained green‐blue were evaluated as positive senescent cells. Cell suspension (1 × 10⁵, 200 μl of serum-free medium) after TGFβ1 pretreatment was seeded onto 8-mm Pore Transwell Inserts (Corning) in 24-well plates with or without matrigel (BD biosciences) and photographed within 24 h.