Xcelligence real time cell analyzer

Cell viability assay was carried out by plating 3000 cells/well into 96-well plates and incubated overnight. The cells were drugged with AZD9291 across a five-concentration range from 10 nM to 1000 nM and then incubated for 72 h [37 (link)]. Viability was assayed by MTT, trypan blue staining, or by a xCELLigence real-time cell analyzer (Roche Life Science, Pleasanton, CA, USA) according to the manufacturer’s instructions [38 (link)].

PC-9 cells (4×10⁴/100μl/well) plated in 96-well plate were placed in 96-well plates and treated with 2 μM of Gracillin, 1 μM of Polyphyllin I, 1 μM of Polyphyllin II, 4 μM of Polyphyllin III, 32 μM of Polyphyllin VI, 1 μM of Polyphyllin VII and 1 μM of Polyphyllin H. Cell viability was then measured using the Cell Counting Kit-8 (CCK-8) to determine cell viability at 24, 48, and 72 h.
Cell viability of human lung adenocarcinoma (PC-9) cells (3×10³/100 μl/well) plated in specific xCELLigence-E plates was sequentially and in real time monitored for 72 h using the xCELLigence-RealTime Cell Analyzer (Roche Applied Science, Mannheim, Germany).

For proliferation assay, the number of cancer cells was monitored by the real-time cell analysis (RTCA) of a cell culture system as described in our previous study [23 (link)]. Briefly, 2.5 × 10⁴ cancer cells were added into an individual well of culture E-Plate (Roche Applied Science, USA) containing 100 μl normal or cancer-associated hMSC-conditioned media. The E-Plates were incubated at 37°C and monitored on the RTCA system by the xCELLigence real-time cell analyzer (Roche Applied Science, USA) at 5-minute time intervals for 7 days. The number of cancer cells in each well was continuously monitored throughout the entire culture period and reported as the cell index. Cancer cells cultured in DMEM+10% FBS served as controls.
For migration assay, hMSC-conditioned media were used to induce migration of hepatoblastoma C3A cells through 8 μm transwell (Costar, Corning, USA) as described in our previous study [24 (link)]. 1 × 10⁶ C3A cells were seeded in the transwell inserts which were placed into wells of the 24-well plate (Costar, Corning, USA) containing 600 μl of various hMSC-conditioned media. After 6 hours of culture, the numbers of C3A cells that migrate to the other side of the transwell's membrane were determined by hematoxylin staining. C3A cells cultured in C3A-conditioned medium served as controls.

The cell proliferation capacity was monitored with an xCELLigence real-time cell analyzer (Roche Life Science, Indiana, USA) according to the manufacturer’s instructions.

xCELLigence Real-Time Cell Analyzer (Roche Applied Science) was used to measure the changes in migration and invasion ability of LSCC cells in response to the changes in miR-744-3p or PDCD4 levels. CIM-Plate 16 was adopted for the migration and invasion assay. The dynamic changes of cell number (expressed as cell migration index) from upper chamber (coated with fibronectin) to lower chamber due to the migratory capacitance of LSCC cells were measured continuously over 24 hours. For the measurement of invasion propensity, the upper chamber of CIM-Plate 16 was coated with Matrigel (1:30 dilution; BD Biosciences) for 4 hour at 37°C before cell seeding. Subsequently, the dynamic invasion of LSCC cells through the Matrigel layer was recorded and expressed as cell invasion index.

Huh7 or HepG2 cells were seeded at 3 × 10³ per well in 96-well E-plates and cultured in DMEM in the presence or absence of various concentrations of HGK. The cell proliferation rates were monitored with an xCELLigence real-time cell analyzer (Roche Life Science, Indianapolis, IN, USA) according to the manufacturer’s instructions.