Xcelligence real time cell analyzer dp system

The xCELLigence Real-Time Cell Analyzer (RTCA) DP system (ACEA Biosciences, San Diego, CA, USA) was used to detect the effect that LINC00958 had on the proliferation and invasion of the ESCC cell lines. Forty-eight hours after transfection, the cells were collected. To detect the invasion of the cells, 168 μL of DMEM with 10% FBS was added to the lower chamber of a CIM-Plate 16 (ACEA Biosciences), and 30 μL of DMEM without FBS was added to the upper chamber. The reference value was measured after the CIM-Plate was placed at 37°C for 1 hour. The cells were seeded in a CIM-Plate 16 at a density of 30,000 cells/well. The medium in the upper chamber was supplemented to 100 μL, and performed for 24 hours; the invasion data was tested at regular time intervals. To determine the proliferation of the cells, 50 μL of DMEM with 10% FBS was added to an E-Plate 16 (ACEA Biosciences) to measure the reference value. The cells were seeded in the E-Plate 16 at a density of 2,000 cells/well. The medium in the upper chamber was supplemented to 200 μL, and performed for 72 hours; the proliferation data was tested at regular time intervals.

Cytotoxicity was monitored by the xCELLigence Real-Time Cell Analyzer (RTCA) DP system (ACEA Biosciences, USA) as described [50 (link)]. First, the background of the E-plates was determined in 50 μl of medium, and subsequently, 100 μl of the HEI-OC1 cell suspension was added (1.3 × 10⁴ cells per well). Cells were incubated for 30 min at room temperature, and E-plates were placed into the RTCA station. Cells were grown for at least 24 h, with impedance being measured every 15 min. After the designated treatments, cells were monitored again every 15 min until the end of the experiment. The electronic readout, cell-sensor impedance induced by adherent cells to the electron flow, is displayed as an arbitrary unit, known as the cell index. The normalized cell index was calculated by the RTCA software at the selected normalization time point, which was chosen as the time immediately before the addition of treated drugs. Each treatment was performed in triplicate.

To evaluate the effect of the CXCL9-derived peptides on growth factor-induced endothelial cell migration, the xCELLigence^® real-time cell analyzer (RTCA DP) system (ACEA Biosciences, Inc.; San Diego, CA, USA) was used. First, 160 µL MCDB131 medium (Gibco) supplemented with 0.4% (v/v) FCS (control medium) or 160 µL control medium with 30 ng/mL FGF-2, 10 ng/mL VEGF165 or 10 ng/mL EGF were added to the lower chamber of a cell invasion/migration (CIM)-Plate (ACEA Biosciences, Inc.). After assembly of the lower and upper chamber, 50 µL of serum-free MCDB131 medium was added in the upper wells. Following equilibration of the plate at 37 °C for 1 h, HMVECs were added in the upper chamber at 4 × 10⁴ cells in 100 µL/well. CXCL9(74-103) or CXCL9(86-103) was added together with the cells in the upper compartment at a concentration of 0.3 or 3 µM. The assembled CIM-Plate was left at room temperature for 30 min to allow the cells to settle onto the membrane. Finally, the plate was placed in the instrument at 37 °C to monitor cell migration for 15 h. Cell migration from one compartment to the other was recorded (one recording every minute) as changes in electrical impedance. These changes were converted into cell indices, as a measure of cell migration.

These assays were performed as described previously by using the xCelligence Real-Time Cell Analyzer (RTCA) DP system (ACEA Biosciences, Inc., San Diego, CA, USA), according to the manufacturer’s protocol. Briefly, the MDA-MB-231 or MDA-MB-468 cells (5000 cells in 150 µl medium/well; E-plate) were seeded into each well^{26 (link)}. Subsequently, the impedance of each well of the E-plate was measured continuously for 72–96 h at 37 °C with 5% CO₂. For the cell invasion assay, cells were incubated for 4 h in serum free medium and a total of 40,000–50,000 cells were seeded in the upper chamber of CIM-plates, which was coated with Matrigel solution (Corning, 354,234). FBS containing medium was added to the lower chamber of the CIM-plates. The impedance measurement was recorded for 40 h. The results were analysed using RTCA data analysis software 1.0 (ACEA Biosciences, Inc., San Diego, CA, USA). The cell proliferation and cell migration or invasion indices were calculated from 4–5 replicates of independent experiments.

Cells were seeded into a 16-well E-Plate (harboring a high-density gold electrode array to measure electrical impedance) and transfected with control or miR-193b mimics immediately after seeding as described previously. The culture medium was changed 72–96 h post-transfection. Cell proliferation was monitored continuously and recorded as a delta cell index (DCI) every 30 min for 168 hours by the xCELLigence Real-Time Cell Analyzer (RTCA)-DP system (ACEA Biosciences). The DCI was defined as the cell index (CI) at a given time point plus a Delta value. The Delta value is the difference between a reference value (=1) and the cell index at the Delta time point (CI₁ = CI one hour post-transfection): DCI = CI + (1−CI₁).