Xcelligence rtca dp analyzer

Cell proliferation was monitored by the xCELLigence RTCA DP Analyzer (Roche) for at least 48 h after treatment, following manufacturer’s indications. This apparatus makes it possible to follow the cellular response to treatment in real-time using electrical impedance as the readout. The continuous monitoring of cell viability by the xCELLigence system allows us to distinguish between cell death and reduced proliferation [29 ]. Cells (5 × 10³ cells/well) were seeded into E-plates 16 (Acea Biosciences Inc.) and impedance was continuously recorded in 15 min intervals until the end of the experiment. Cell index (CI) values, derived from the measured impedances, were acquired by the RTCA Software V1.2 (Roche) and exported to Microsoft Excel for normalization of data of each single well to the first measurement after starting the treatment. Statistical analysis and graphical representation of data were performed by the Prism GraphPad Software V5.0 (GraphPad Software Inc., La Jolla, CA, USA). Data displayed in the graphs is the average value of three biological replicates, each consisting of two technical replicates.

Cell activation was characterized in real time by using an xCELLigence RTCA DP analyzer (Roche Diagnostics, Pleasanton, CA, USA). BV2 cells were seeded at 2.5 × 10⁴ cells into each well of an E-Plate 16 (Roche Diagnostics, Pleasanton, CA, USA). After 24 h of initial adherence, BV2 cells were treated with IFN-γ (10 ng/mL) and LPS (100 ng/mL) and GYY4137 (200 μM) or DMSO (solvent control). Impedance measurements were recorded every 15 s during the first 2 h of cultivation and then every 15 min overnight. Impedance measurements were recorded as a combined cellular index of proliferation, viability, activation, and morphology changes.