Xcelligence rtca dp real time cell analyzer

First, the epithelial barrier integrity was determined by measuring electrical impedance using xCELLigence RTCA DP Real-Time Cell Analyzer (ACEA Biosciences, California, USA). Caco-2 cells were seeded on RTCA E-plates (E-plate 16) at a density of 10⁵ cells/well. We applied the control (CTRL) and H₂O₂ or H₂O₂+olaparib treatment groups. After attaining confluency, the monolayers were treated with different concentrations of H₂O₂ (100, 200, 500, and 1000 μM) or with olaparib (10 μM) as a pretreatment, 30 min before H₂O₂. The CTRL and H₂O₂ treatment groups received the same amount of DMSO as the olaparib-treated cells. The cell index (CI) was continuously monitored by the equipment for 24 hours.

Briefly, the culture medium (50 µL) was pipetted into a special E-plate with gold electrodes at the bottom of the wells and the background impedance was determined. The cell attachment and proliferation were measured as a change of impedance between electrodes and expressed as a unitless cell index. The appropriate number of cells (5000 rMSCs or 2500 C6 cells per well) was added in the culture medium (100 µL) and the cells were allowed to attach to the bottom of the wells for 2 h. This was followed by the addition of nanoparticles or their leaches in the medium (50 µL); the final particle concentration was kept at 20 µg/mL per well. The plates were then inserted into the xCELLigence RTCA DP real-time cell analyzer (ACEA Biosciences, now Agilent Technologies; Santa Clara, CA, USA) and the changes in impedance were recorded every 20 min for 3 days. All experiments were done in triplicates and repeated three times.