Xcelligence real time cell analyzer dp instrument

To monitor the proliferation and migration of A549 cells in real time, the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument (Roche Diagnostics GmbH, Germany) was used in this study. The relative impedance change (cell index) of the microelectronic sensor that was integrated into the bottom of the well was used to measure the proliferation and migration of A549 cells. For proliferation assay, A549 cells were seeded in E-plates 16 (Roche Diagnostics GmbH) at 5000 cells/well. HSV-PDGFi-shRNA or vector was added after the cells adhered to the wall overnight. Then, the plates were continuously monitored once every 15 min for 72 h. For the cell migration assay, the migration of A549 cells was assessed using specifically designed CIM-plates 16 (Roche Diagnostics GmbH), in which the upper and lower chambers of each well were space out by an 8-μm microporous membrane containing matrigel. The 10% FBS medium was added in the lower chambers, and the cells were seeded into the upper chambers at 40,000 cells/well in serum-free medium. The CIM-plate 16 was monitored every 15 min for 72 h in total. Data analysis was carried out using RTCA software 1.2 supplied with the instrument.

To quantify cell growth, background hindrance was quantified in culture medium per well as described earlier [32 (link)]. Later HCT-116, SNU-C2A, and HT-29 cells were seeded on 16-well E-plates and incubated in Roche xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche Diagnostics GmbH, Germany). TMP (10 μM)- and TGFβ (10 ng/mL)-treated cell index values were measured every 15 min time intervals.

Invasion activity of cells were determined by xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche Diagnostics GmbH, Basel, Switzerland). Top chamber (invasion/migration (CIM)-Plate 16) was pre-coated with matrigel (BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA) for 4 h, add only 10% FBS containing medium to under chamber then chambers were assembled. Serum-free medium was previously measured then A549, DU145, and MIA PaCa-2 cells were seeded on top chamber with matrine (50 μM). Cell index was measured every 15 min time intervals [77 (link)].

Real-time cell proliferation was measured using the xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument with an E-plate (Roche Applied Science, Penzberg, Germany). After running the background blank with 100 μL complete culture medium, the cells were seeded in wells, and the cell proliferation was continuously monitored as the relative rate of change (cell index) every 30 min. All data were recorded using RTCA software. For the SRB assay, 7000 T47D cells were seeded in a 96-well plate. After a specific treatment period, cells were fixed with 10% trichloroacetic acid (TCA) overnight at 4 °C. Then, cells were washed with 1% acetic acid and stained with 0.04% SRB dye for 10 min at room temperature. SRB dye was released by adding 10 mM Tris-base solution, and optical density was measured by a microplate reader at 515 nM.

To monitor cell migration/invasion in real time, we used the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument equipped with a CIM-plate 16 (Roche, Indianapolis, IN), which is a 16-well system in which each well is composed of upper and lower chambers separated by an 8-μm microporous membrane. Migration/invasion was measured as the relative impedance change (cell index) across microelectronic sensors integrated into the bottom side of the membrane. For cell migration experiments, cells (7.5 × 10⁴ /well) were added in duplicates to the upper chambers. MCF-7 breast cancer cells were treated with flavone (88 μM), apigenin (30 μM) and luteolin (43 μM). Migration/invasion was monitored every hour for 9 h. For quantification, the cell index at the indicated time points was averaged from three independent measurements.