T 25 tissue culture flasks

Human glioblastoma cell line, U87MG, maintained in MEM (GIBCO, USA) supplemented with 10% FBS (GIBCO, USA), 500 U/ml penicillin (Sigma, USA), and 200 mg/L streptomycin (Biowest, USA) was obtained from Pasteur Institute of Iran (Tehran, Iran). Cells were cultured as a monolayer at a density of 200,000 cells/ml in T-25 tissue culture flasks (SPL, USA) in an incubator (MEMmert, Germany) in 5% CO₂ humidified atmosphere at 37°C. To achieve 70-80% of confluency and ensure to maintain cells in the logarithmic phase of growth, cells were Trypsinated (1 mM EDTA/0.25% Trypsin (w/v), Sigma, USA) and then sub-cultured. Cell viability >97% was confirmed by trypan blue staining. Spheroids of uniform size and diameter from 450 to 550 µm were produced using the liquid overlay technique. U87MG cells (5×10⁵) were seeded in 100-mm dishes coated with a thin layer of 1% agar (Bacto Agar, Difco, Detroit, MI, USA) in 10 ml MEM supplemented with 10% FBS and incubated in the above described condition for one month. Half of the culture medium was replaced with fresh medium twice per week.

Human fibrosarcoma (HT-1080) and colorectal adenocarcinoma cells (HT-29) were obtained from the Korean Cell Line Bank (Seoul, Korea). Typically, the cells were cultured in T-25 tissue culture flasks (SPL, Korea) in RPMI-1640 media containing 10% FBS and 1 × P/S and incubated at 37 °C in a humidified incubator with a 5% CO₂ atmosphere. When the cells reached 80% confluence, the culture media were changed to fresh serum-free conditioned media. For immunoblotting and zymography, the media were collected 48 h after culture and concentrated by using an Amicon ultra centrifugal filter unit (50 kDa MWCO, Millipore, Billerica, MA, USA) and by centrifugation (1500× g for 20 min).