Xcelligence sp system

In order to gain detailed information concerning the effectivity of the hybrid molecules during the proliferation of the neuroblastoma cells, real-time neuroblastoma cancer cell proliferation was determined using the xCELLigence SP system (Roche Applied Science, Mannheim, Germany). In contrast to the MTS assay, the xCELLigence assay allowed us to follow the growth of cells continuously in the absence and presence of the added drugs.
Neuroblastoma cells were seeded in 96-well plates (E-Plate 96, ACEA Biosciences, San Diego, CA, USA), 20,000 cells/well. Real-time dynamic cell proliferation was monitored in 30 min intervals for 96 h at 37 °C. 24 h after seeding, and cells were treated with M and the MBG hybrids, as indicated. Cell index values were calculated using the RTCA Software (2.0). All curves were normalised to the time point after drug treatment was conducted (~24 h after seeding) applying the RTCA software [32 (link),33 (link)].

The xCELLigence SP system (Roche Diagnostics GmbH, Mannheim, Germany) was used for the real-time analysis of cell proliferation. This system measures the changes in impedance in specific plates, with micro electrodes covering the well bottoms (E-plates, Roche Diagnostics GmbH, Mannheim, Germany). The relative changes were recorded as the Cell Index, which is a dimensionless parameter. 1 × 10⁴ UM-SCC1 cells were transfected with either siRNA or plasmids and seeded onto a 96-well e-plate according to the manufacturer's instructions. Cells transfected with siRNA were seeded 24 h after the second transfection. Cells transfected with plasmids were seeded 24 h after the plasmid transfection. Cell proliferation was monitored for 120 h, and the data were evaluated using the RTCA 2.0 software (Roche Diagnostics GmbH, Mannheim, Germany). All cell proliferation experiments were repeated three-fold (n = 3), and at least a triplicate of every cell population was analyzed in each experiment.

Stably transfected HEK293 cells were cultured in DMEM/F12 medium containing 10% FBS and 0.8 mg/ml geneticin G418 in a humidified environment with 5% CO₂ at 37°C. Cells were maintained in T25 flasks and passaged thrice a week. For passaging, cells were carefully rinsed with PBS (pH 7.2), detached using 0.05% Trypsin–EDTA, and a fourth of the cell suspension was further cultivated. Cell numbers were routinely determined using the Countess Automated Cell Counter (Invitrogen) according to the manufacturer’s instructions. In addition, cell proliferation was monitored in real time and noninvasively using the RTCA biosensor technology in the xCELLigence SP system (Roche) using 96-well E-plates (Omni Life Science). After thawing, each batch of cells was tested for potential mycoplasma contamination using the MycoAlert Detection Kit and the MycoAlert Assay Control.