Binocular loop

Migration and invasion were determined by a two-chamber transwell assay (Corning Incorporated; Corning, NY, USA). The upper side of the polycarbonate film was left untreated (migration) prior to cell seeding or was covered with Matrigel™ (500 ng/μL; BD Biosciences; Franklin Lakes, NJ, USA) (invasion, chemotaxis assay). 600 μL of complete medium was added into the lower chamber. After succinate addition, 2 × 10⁴ cells were suspended in 100 μL supplement-free medium and added into the upper chamber (inserts). After incubation for 24 h at 37  °C, transwells were gently picked up, invaded cells on the bottom of the inserts were rinsed with PBS. Nucleic acids were stained with 0.05% crystal violet and photographed with a binocular loop (Leica; Wetzlar, Germany) microscope equipped with a Mrc camera (Zeiss; Oberkochen, Germany). Cells were counted using the ImageJ software (NIH; Bethesda, MD, USA).

Cells were seeded at 2 × 10⁴ were plated in the upper chamber with DMEM without FBS complementation to allow migration for 24 h at 37 °C. Normal DMEM medium with 10% FBS complementation was distributed in each well, below the chamber. Chambers were gently picked up before a brief PBS rinse and 0.05% crystal violet coloration. Cells at the bottom position of the chamber were photographed and counted with a binocular loop (Leica).