Transwell co culture chambers

Breast cancer cells (2.0–3.0 × 10⁵) were seeded in the upper well of the Corning Transwell co-culture chambers (Corning, NY, USA). When the cells adhered, the induced mature adipocytes were seeded in the lower chamber. The supernatant from the upper and lower chambers was replaced with DMEM/F12 containing 2% FBS. Subsequent experiments were performed after three days of co-culture.

All cell lines were obtained from the cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cell lines were identified by cell species, STR analysis and mycoplasma detection to ensure no contamination by other cells or foreign microorganisms. Human PCa cell lines PC-3, DU145 and 22RV1 were cultured in DMEM (Gibco, China) containing 10% FBS (Gibco). Mouse embryonic osteoblast precursor cells (MC3T3-E1) were cultured in MEM-a (Gibco) containing 10% FBS (Gibco). All cells were cultured at 37 °C in a humidified environment with 5% CO₂. PCa cells and osteoblasts were co-cultured in a 6-well plate with 0.4 µm Transwell co-culture chambers (Corning, USA). PCa cells were plated in the lower layer of the chamber 24 h before co-culture, and then osteoblasts were seeded in the upper layer of the chamber. The ratio of osteoblasts to PCa cells was 5:1. To provide sufficient nutrition, 3 ml of MEM-a with 10% FBS was added to the lower chamber and 1.5 ml of serum-free medium was added to the upper chamber. The co-culture duration was 3 days, and the medium was replaced every 24 h.

LLC cells were seeded at a density of 70% for the experiment. LLC cells were washed twice with 1XPBS and replaced with fresh medium before co-culture. Next, transwell co-culture chambers with a 0.4um pore size (Corning) were spread above a 6-well plate. LLC-SD cells (1*10^4) were added to the trasnwell chambers and resuspended in F12/B27 medium. After co-culture for 24 h, the upper transwell chambers were removed, and the lower LLC cells were digested with trypsin and used for migration and invasion assay.