6 well transwell insert

HSCs were activated with TGF-β1 (10 ng/ml, PeproTech, United States, CAT: 100-21-10) for 24 h, and then, activated HSCs were cocultured with SD-BMSCs by using 6-well Transwell inserts (0.4 µm, Corning, NY, United States, CAT: 3412), which were permeable to the culture medium but not cells. All coculture experiments were performed with HSCs seeded in the bottom chambers and BMSCs seeded in the insert chambers. After coculture for 12 h, the cells were washed twice with PBS, randomly divided into 5 groups and incubated in fresh DMEM. One group with normal medium was set as the model group. The group with medium containing 6 µg/ml colchicine was used as a positive control. Another three groups with medium with three doses of FA (1 mg/ml, 0.5 mg/ml, 0.25 mg/ml) were set as the FA groups. In addition, inactivated HSCs cocultured with BMSCs were used as the normal group. After coculture for 24 h, we collected HSCs and medium for subsequent experiments.

RAW 264.7 macrophages (American Type Culture Collection, ATCC, Manassas, VA, USA) were seeded in 6-well culture plates (Corning Inc., Corning, NY, USA), at a density of 1.5 × 10⁵ cells/mL, in 3 mL of Dulbecco’s modified eagle medium (DMEM, Sigma-Aldrich Corporation, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, BRL), 1 mM L-glutamine (BioWhittaker Europe, Verviers, Belgium), penicillin (200 µg/mL, BioWhittaker Europe, Verviers, Belgium), and streptomycin (200 µg/mL, BioWhittaker Europe) at 37 °C under a CO₂ (5%) atmosphere. To analyze the effects of the ions released by MBG-75S, concerning the possible regulatory role of macrophages on in vitro osteogenesis, RAW 264.7 cells were cultured in the absence (control macrophages) or in the presence of 1 mg/mL of MBG-75S deposited on 6-well transwell inserts (0.4 µm pore size, Corning Inc., Corning, NY, USA), which is covered by the culture medium. After 24 h of culture, the media from these macrophage cultures were collected and diluted 1:1 with α-MEM for subsequent treatment of MC3T3-E1 pre-osteoblasts (10⁴ cells/mL) for 10 days, as it is indicated in Scheme 1. ALP activity of MC3T3 -E1 cells was then assessed as specific marker of in vitro osteogenesis, as described in Section 2.7.

In the Boyden chamber, 4 × 10⁵ HUVECs in 1 ml of complete media were seeded into the 6-well Transwell inserts (24.5 mm in diameter, 8 µm pore size, Corning) and incubated at 37 °C for two days to allow formation of a confluent monolayer on the top of the porous filter membrane. The HUVEC monolayer was activated by 20 ng/ml TNFα for 4 h, and then rinsed with PBS three times. Subsequently, 4 × 10⁶ cancer cells in 1 ml serum-free media were added to the insert (top chamber), and then 2 ml of conditioned media, or 20 ng/ml S100A8/A9, or 100 ng/ml CXCL12, or 40 ng/ml CCL2, or 0.1% FBS were added to the reservoir plate (lower chamber). Cancer cell chemoattractants were diluted in serum-free media. Two days later, transmigrated (TEM⁺) cancer cells were detached by immersing the reservoir side of the insert in cell dissociation buffer (enzyme-free) for 1 h at 37 °C and then collected.