Xcelligence rtca system

The effect of different concentrations of copper on Caco-2 proliferation was determined using real-time cellular analysis (RTCA). After 24 h stimulation, cells (8 × 10³ cells/well) were seeded in several E-Plate 16 dishes (ACEA Biosciences Inc., CA, United States) for proliferation assays. The plates were kept in the cell incubator at 37°C with 5% CO₂ for 3–4 days. The cell index and growth curves were automatically recorded on the xCELLigence RTCA System (ACEA Biosciences Inc., CA, United States).

Cell proliferation was assessed using the xCELLigence RTCA system (Acea Bioscience, San Diego, CA, United States, distributed by Roche Diagnostics) that allows long-term monitoring of live cells in a noninvasive manner (Heinecke et al., 2014 (link); Al Nakouzi et al., 2016 (link)). In brief, 5,000–10,000 cells were seeded in each well of E-16-well plates (Roche). Cell proliferation was monitored for 40–70 h at 37°C in the incubator. Microelectrodes on the bottom of plates were used to detect impedance changes proportional to the number of adherent cells. The impedance value of each well was automatically recorded by Real-Time Cell Analyzer (RTCA) software. Two parallel wells were included for each sample in one replicate, and three independent replicates were conducted.

Proliferation was performed using xCELLigence RTCA System (Acea Biosciences). Briefly, 2000 primary mouse ECs per well were seeded in gelatin-coated E16 plates in 2% FBS, allowed to adhere for 7 h and then stimulated with growth factors (VEGFA₁₆₅ 200 ng/ml or FGF2 100 ng/ml) or vehicle (PBS). Proliferation curves show ~60 h of monitoring with readings of cell index performed every 15 min. Cell index values at 24 h were used to calculate fold changes in proliferation of indicated growth factor versus vehicle. ECs migration was assessed using an in vitro wound healing assay as previously reported^{47 (link)}. Briefly, cells were seeded on Ibidi-culture inserts (Ibidi, #80,209) to create a wound between two adjacent EC monolayers. At confluency, insets were removed, and cells allowed to migrate. Pictures of wound width were taken before and after stimulation (8 h) and % closure was calculated.

An xCELLigence RTCA system (ACEA Biosciences, San Diego, CA, USA) was used to determine the impact of HDV innate immune recognition on cell viability. HepG2-NTCP cells were co-cultured with genetically modified HBV-specific T-cells [25 (link),26 (link)] and T-cell induced antigen-specific killing rates were measured. Therefore, HepG2-NTCP and HepG2-NTCP MDA5 ko cells were differentiated for 14 days. Differentiated cells were co-infected with either HBV and HDV or only HBV and infection was established for seven days. Afterwards, infected cells were seeded at a density of 5 × 10⁴ cells/well on collagenized xCELLigence 96-well plates and rested for two more days prior to start of co-culture. T-cells were added to seeded cells in different effector (T-cell) to target (HepG2-NTCP cell) ratios (1:1, 1:3, 1:9). Cell viability was determined as cell index and normalized to the start of co-culture.

For in vitro expansion edited T cells were incubated with EBV-transformed B cells (LCL) in a 1:7 (E:T) ratio and the described protocol was performed accordingly^{33 (link),34 (link)}. CAR frequency and absolute numbers were monitored and measured over indicated time points of expansion.
Sequential stimulation experiment was performed using expanded and rested engineered CAR T cells. Expamers stimulation was done as previously described. Stimulation signal was disrupted by adding 1 mM Biotin. Cells were subsequently washed and transferred into fresh media containing low dose IL-2 (25 IU ml⁻¹). Surface expression of activation markers was monitored by flow cytometry (see below) and cell count was measured (Nucleocounter, Chemotec).
For in vitro killing assay, xCELLigence RTCA System (ACEA Biosciences Inc.) was used. Specific lysis was measured targeting CD19 expressing Human Embryonic Kidney cells (HEK293-CD19⁺). 2 × 10⁴ target cells were seeded into 96 well E-Plate (ACEA Biosciences Inc.) and rested overnight. Engineered CAR T cells were added in a 5:1 (E:T) ratio and incubated for indicated time. Changes in impedance signal were measured in real-time and analyzed using RTCA Software Pro (ACEA Biosciences Inc.). All used cell lines were unmodified and purchased from ATCC.