Rtca dp instrument

NCI-H460 and A549 cells were trypsinized briefly until detached. Cells were resuspended in complete growth media and counted. Optimal cell number per well (5000) was determined in initial titration experiments. According to xCELLigence (Roche) manufacturer’s instructions, cells were seeded in duplicates into E-plates 16 (Roche, cat# 05469830001) after baseline measurement. Plates were incubated for 1 hour at room temperature, and then placed into the RTCA DP instrument (Roche, cat# 05469759001) located in an incubator preserving same temperature and CO₂ concentration as were used for routine cultivation of the cells. siRNA transfection mix was added to the cells 6 hours after seeding, and left there for 4 hours. After that, transfection mix was replaced with regular growth media. Cell index (arbitrary unit reflecting the cell-sensor impedance) was measured every 15 minutes during the first 4 hours for better resolution at attachment and spreading phase. Further measurements were taken every 30 minutes. Doubling times were calculated with RTCA software 1.2 (Roche). At least three independent experiments were performed.

Cell proliferation and migration assays were performed using the xCELLigence System (Roche Diagnostics GmbH) with RTCA DP Instrument (Roche Diagnostics GmbH) according to the manufacturer’s protocol. When the cells entered the logarithmic growth phase, culture medium was aspirated and replaced with fresh culture medium ± HMGB1 at the concentration of 10–200 ng/ml. Cell proliferation and migration were continuously monitored every 5 min using the RTCA DP Instrument via calculation of the “cell index” (to reflect the surface area covered by the cells) for each plate. Each sample was evaluated in triplicate.

The xCELLigence Real-Time Cell Analyser (RTCA) DP Instrument equipped with a CIM-plate 16 (Roche) was used as previously^{42 (link)}. For quantification, the cell index at indicated time points was averaged from three independent experiments.

To analyze endothelial cell migration using the CIM-Plate 16, human endothelial cells EA.hy926 were cultured in DMEM with high glucose supplemented with 10% FBS; cells were deprived in serum-free DMEM for 6 to 8 h prior to the experiment and seeded 30 × 10⁴ cells in 50 uL of serum-free DMEM per well into the upper chambers of the CIM-Plate 16. Samples of MSC-CM (160 uL per well) were placed into the lower chambers. DMEM supplemented with 10% FBS was used as a positive control and serum-free DMEM served as a negative control. Then CIM-Plate 16 was placed in the RTCA DP Instrument (Roche, Basel, Switzerland) equilibrated in a CO₂ incubator. Endothelial cell migration was continuously monitored using the RTCA DP Instrument. MSC-CM provided a strong chemoattractant signal, which induced the directional migration of endothelial cells through the micropores of the CIM-Plate 16. Migrating cells were detected by the electronic sensing microelectrodes, producing changes in the measured Cell Index values. Time-dependent cell migration was monitored over 4 h. All experiments were performed in duplicates. The RTCA Software 1.2 was used to calculate Cell Index values for MSC-CM-mediated endothelial cell migration.

The E-Plate 16 was pre-equilibrated with 100 μl of medium in the tissue culture hood for 0.5–1 h and then 2 × 10³ of cells resuspended in 100 μl of medium were added, and kept in the culture hood for another 30 min. The real-time recording of proliferation was carried out on the RTCA-DP instrument (Roche) and monitored every 1 h for 3 days.