Dulbecco s modified eagle s medium

The Transwell experiment, which evaluated the migration ability of Y79 cells in various groups, was performed. Incubation medium (with 1 mM MgCl₂; 200 µl) containing 1×10⁴ cells was seeded into the upper Transwell chambers (Costar; Corning Incorporated, Corning, NY, USA). The cells were subsequently incubated at 37°C for 48 h to allow cell migration through the porous membrane (pore size, 8 µm). Upon completion of the culture, cells remaining on the upper surface of the chamber were completely removed using a cotton swab. The lower surfaces of the membranes were fixed with 4% paraformaldehyde for 20 min and stained in a solution containing 0.5% (w/v) crystal violet for 5–10 min at room temperature. Subsequent to being washed with ddH₂O, results of different groups were observed using the Olympus CX41 microscope (Olympus Corporation, Tokyo, Japan) at ×200 magnification and the numbers of cells were determined using Image-Pro Plus 6.0 software (Nikon Corporation, Tokyo, Japan). The invasion ability of Y79 cells was then measured as aforementioned, with polycarbonate membranes pre-coated with 100 µl Matrigel (in 0.8 µg/µl Dulbecco's modified Eagle's medium; BD Biosciences, San Jose, CA, USA) at 37°C for 2 h to form a reconstituted basement membrane.

MKN45 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were cultured at 37°C in 5% CO₂ atmosphere in appropriate tissue culture medium and passaged for no longer than 2 months. Cells were used for experiments (in vitro and in vivo) when they reached ~80% confluence.
In our study, MKN45 cells were used to generate xenograft tumor model on nude mice. All the animal studies were conducted in compliance with the guidance for the care and use of laboratory animals and were approved by the Life Science Research Ethics Committee of the Institute of Southwest Hospital, Third Military Medical University (Chongqing, People’s Republic of China). Nude mice were obtained from the Southwest Hospital Animal Center of the Third Military Medical University. For the animal tumor models, 6- to 8-week-old female nude mice were selected, and tumors were established by subcutaneous injection of 1×10⁶ MKN45 cells resuspended in 100 μL of 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) into the rear flank of mice. The tumor sizes were monitored every 2 days, and the animals were subjected to in vivo experiments when the size of tumors was over 5 mm (about 15 days after inoculation).

MG63, U2OS, and 143B were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). Human bone marrow mesenchymal stem cells (hMSCs), Saos-2, and HOS were purchased from Procell Biological Technology (Wuhan, China). Saos-2 cells were cultured in McCoy’s 5a medium (Procell Biological Technology) supplemented with 15% fetal bovine serum (FBS; BI, Kibbutz Beit Haemek, Israel). U2OS cells were grown in Dulbecco’s modified Eagle’s medium (BD Biosciences, Franklin Lakes, NJ, USA) containing 10% FBS (BI). 143B cells were cultured in Eagle modified essential medium (Shanghai Zhong Qiao Xin Zhou Biotechnology) supplemented 10% FBS (BI). MG63 and HOS cells were grown in minimum essential medium (Gibco, Grand Island, NY, USA) containing 10% FBS (BI). hMSCs were cultured in hMSC complete medium (Procell). All cell lines were maintained in an incubator at 37°C with 5% CO₂. Cisplatin (DDP) was obtained from Meilun Biotechnology (Dalian, China). In experiments using cisplatin, cells were incubated with 5 μmol/L DDP for 24 h before the detection.