Xcelligence real time cell analyser

Cell viability was assessed using the xCELLigence real-time cell analyser (ACEA Bioscience, Agilent, San Diego, CA, USA). siRNA-transfected B4G12 cells were seeded at 4 × 10⁴ in triplicate in an E-Plate 96 (ACEA Biosciences) and left to attach for 18 hrs. Cells were left untreated or treated with indicated compounds. The electrical impedance readings of overall cell viability were recorded, using the xCELLigence real-time cell analyser, throughout the experiment for at least 8 h. The percentage of viable cells was calculated for each time point relative to time zero, which was set as the last impedance reading prior to addition of compounds. Data shown are representative of three independent experiments. Statistical analysis was performed using GraphPad software (Prism, version 7, GraphPad, San Diego, CA, USA). Groups were compared with two-way ANOVA, followed by post-hoc Bonferroni test, for multiple comparisons, with p-value < 0.05 considered significant.

Continuous cell proliferation of NCI-H460 and NCI-H460/R cancer cells untreated and treated with DOX after 24 h from initial seeding (10, 50, 100, 500 and 1000 nM) was analysed using the xCELLigence Real Time Cell analyser (ACEA Biosciences Inc., USA) which facilitates label free real-time cell analysis by measuring impedance-based signals across a series of gold electrodes. Using E-plates, 50 μL of complete medium, RPMI 1640 was added to each well and the electrodes were allowed to stabilise for 30 min. The plates were then moved into the xCELLigence Real Time Cell analyser to set a base line without cells. The cells were then seeded on an E-plate at the following densities: 4.0 × 10³ and 8.0 × 10³ cells in a 200 μl medium per well. Cells on the electrodes were monitored by reading and recording the cell impedance every 30 min through 338 sweeps.

Analysis was performed on xCELLigence real time cell analyser (RTCA) (ACEA Biosciences) measures focal adhesion of living cells in real-time50 (link). Cells were seeded onto custom RTCA E-plates (ACEA Biosciences) coated with high-density gold arrays for measuring electrical impedance. The xCELLigence biosensor measures cellular adhesion, which is converted to Cell index (unit less) by the xCELLigence software (version 1.2.1). Cells were seeded into E16 well plates and allowed to recover until the cells had attained a stable Cell index. Treatment of cells on the plates with Aβ(1-42) was performed in serum free DMEM. After 4 h of treatment inactivated FBS (10%) was added into plates.