Evaluating Proliferation and Invasion of Cervical Cancer Cell Lines

To evaluate the proliferative ability of CC cell lines, C-33A, SiHa and HeLa cells were seeded (1 × 10⁴ cells/well) into 400 µL of medium in an E-plate L8 device (iCELLigence system, ACEA Biosciences, San Diego, CA, USA), after measuring the background in 100 µL of medium. Two replicates for each condition were analyzed. Cell attachment, spreading and proliferation were monitored by real-time cell analysis for 7 days, on the basis of changes in cell-sensor impedance, as previously described [47 (link)]. To assess the invasive capability of CC cell lines, cells were starved overnight. Next day, extracellular matrix layer was prepared by adding 40 µL of Matrigel^® (BD Biosciences, Franklin Lake, NJ, USA) into Transwell^® inserts with 8-μm-pore membranes (Sarstedt, Nümbrecht, Germany). After gelling for 15–30 min at 37 °C, starved cells were seeded (1.25 × 10⁵ cells/insert) on the gel layer in FBS-free medium, and the insert was placed in a well with FBS-containing medium. Cells were allowed to digest and penetrate the Matrigel^® layer as far as the membrane for 72 h. Then, non-invading cells and the gel layer were removed, and membranes were fixed and stained with a crystal violet solution containing paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA). Images were taken with a Leica DMi1 microscope and the Leica Application Suite v4.12 program (Leica, Wetzlar, Germany).