Xcelligence rtca dp

Evaluation of Am cytotoxicity was conducted based on extract toxicity assay according to ISO-10993 norm. Cytotoxicity of AM extracts were evaluated using MTT Cell Proliferation assay and real-time cell analyser (xCELLigence RTCA DP, Roche Applied Science, Germany). Am (∼6 cm²) was extracted in DMEM/Ham’s F12 media (Sigma, Germany) containing 10% FBS (Sigma, Germany), 10 ng/ml bFGF (GIBCO, USA), 5 μg/mL amphotericin B (Sigma, Germany), 100 μg/mL streptomycin (Sigma, Germany) and 100 U/mL penicillin (Sigma, Germany) for 24 h (36°C, 5% CO₂). After 24 h exposure to different concentrations (100%, 50%, 25% and 12,5%) of Am extract the number of live cells per well was determined by absorbance measurement at 570 nm. MSC viability was presented on histogram as an average from five measurements. To evaluate cytotoxicity of Am extracts using a real-time cell analyser MSC were seeded on E-Plates 16 (5×10³ cells/well) and cultured until reaching a log- phase growth in standard medium. After 24 h incubation, the same concentration of Am extracts were added to the wells. Cells were exposed to Am extracts for 72 h. Results were presented on a graph as an average from five measurements.

The xCELLigence RTCA DP (Roche) instrument was used to perform the real-time cell proliferation assays according to the manufacturer’s instructions. The concomitant changes in Cell Index reflected the changes in cell numbers. For colony formation assays, 1 × 10³ cells were seeded in a well of a 6-well plate and cultured for 1–2 weeks. The cells were then fixed and stained. The cell colonies were imaged and analyzed.

The real-time analysis (growth, migration, and invasion) of TNBC cells was performed by using real-time cell analyzer (xCELLigence RTCA DP, Roche, Penzberg, Germany) [10 (link), 13 (link), 17 (link)]. Based on the electric impulse generated in the gold particle of E-Plate and CIM-plates of the analyzer, the real-time data on growth, migration, and invasion were obtained.

The real time analysis of cell migration, growth index and invasion were performed by using a real time cell analyzer (xCELLigence RTCA DP, Roche, Germany), as previously described in detail [35 (link), 77 (link)].

A real time cell analyzer (xCELLigence RTCA DP, Roche, Penzberg, Germany) was used for the real time analysis of cell migration, invasion and growth index [14 (link),15 (link)]. 100 μL of cell suspensions (5 × 10⁴ cells/mL) were seeded on each of the 16 well E-plate for cell growth index. CMI plates were used for the analysis of cell migration and invasion, where the lower chamber wells were filled with chemotaxis inducer (10% serum supplemented media), and 100 μL of cell suspensions (5 × 10⁴ cells/mL) in serum free medium were added into the wells of upper chamber. For cell invasion assay, the membrane of the CMI plate was pre-coated with Matrigel (354277, BD Biosciences, Sparks, MD, USA) with 1:40 dilution in 1× PBS before cells were seeded. After a certain period of cell growth (usually 4 h, indicated in the figures), TQ of different concentrations (1–10 μM) were added into the wells. The process of cell migration and invasion was monitored every 30 min till the experimental endpoint.