The protocol for the migration assay with the xCELLigence RTCA‐DP system (Roche) was described previously [45 ]. Briefly, cells were serum starved for 4–6 h, then detached with trypsin, and resuspended in serum‐free medium with concentration of 1 × 105 cells·mL−1. 100 μL of suspension was seeded into the pre‐equilibrated upper chamber of the CIM plate, and the low chamber was added complete medium for migration. Cell index values were detected every 15 min following procedure. RTCA software v1.2 (Roche Applied Science, Indianapolis, IN, USA) was used to calculate the slopes of the curves at various time points.
Rtca software v1
Manufactured by Roche
Sourced in United States
The RTCA software v1.2 is a software package designed for the analysis and monitoring of real-time cell-based assays. It provides a user-friendly interface for data acquisition, analysis, and visualization of cellular responses in real-time.
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7 protocols using rtca software v1
1
Migration Assay with xCELLigence RTCA-DP
2
Real-Time Cell Proliferation Monitoring
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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.
3
Monitoring Melanoma Cell Responses
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Background was measured with 50 µL medium per well in an E-Plate 16 (Roche). Then 3×103 or 5×103 cells/well were seeded in 150 µL additional volume. Cell attachment was monitored with RTCA SP (Roche) instrument and the RTCA Software V.1.2 (Roche) every 10 min. After 16–20 hours, melanoma cells were transfected with 100–200 ng/mL 3pRNA or Ctrl RNA and incubated for 10–15 days at 5% CO2, 37°C. As additional controls, cells were treated with Lipofectamine 2000 only or left untransfected. Six hours post-transfection, 10% FCS was added to Ma-Mel-86c and Ma-Mel-47 cells, the medium was exchanged entirely for UKE-Mel-164a cells. All experiments were carried out as triplicates. Changes in the electric impedance were given as a dimensionless cell index value derived from relative impedance changes corresponding to cellular coverage of the electrode sensors. The cell index was first normalized to the baseline impedance value of the background and then to the time point of transfection.
4
Pancreatic Cancer Cell Migration Assay
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The migration assay was performed with the xCELLigence RTCA-DP system (Roche) as previously described methods with some modifications34 (link). Briefly, 6 × 104 serum-starved pancreatic cancer cells were resuspended in 100 μL of serum-free medium which was added with PMA (30 ng/mL, final concentration) and a series of concentrations of JYQ-42 and then were added to the pre-equilibrated upper chamber of the xCELLigence CIM plate along with the bottom well plate containing complete medium which was added with PMA (30 ng/mL, final concentration), IL8 (10 ng/mL, final concentration) and a series of concentrations of JYQ-42 for migration. Cell index values were recorded every 15 min during the process. RTCA software v1.2 (Roche Applied Science) was used to calculate the slopes of the curves at various time points.
5
Real-time cell migration assay
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The procedure was carried out as previously described [20 (link), 26 (link)]. Briefly, 4 × 104 of each of serum-starved DU145 stable cells were suspended in 100 μl of serum-free medium, and then cell suspension was added into the pre-equilibrated upper chamber of CIM-plate. The lower chamber was filled with 160 μl of complete DMEM containing 10% FBS. The kinetic cell index of migration was recorded every 15 min for 24 h and then calculated by RTCA software v1.2 (Roche Applied Science).
6
Real-Time Cell Analysis of Migration, Invasion, and Proliferation
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RTCA analysis for cell migration, invasion and proliferation was performed according to a routine protocol [22 (link)]. For migration and invasion assays, 1 × 105 serum-starved Ishikawa and SPEC-2 cells treated with peptides or peptidomimetics were resuspended in 100 μl of serum-free medium and added to the preequilibrated upper chambers of an xCELLigence CIM plate, and the bottom wells of the plate contained complete medium. In parallel, 2 × 103 serum-starved Ishikawa and SPEC-2 cells were resuspended and loaded into the upper chambers of an xCELLigence CIM plate for proliferation analyses. Cell index values were detected every 30 min for 30 h and 100 h throughout the migration/invasion and proliferation assays, respectively. We used RTCA software v1.2 (Roche Applied Science) to calculate the slopes of the curves at various time points.
7
Real-Time Cell Analysis of A549 Cells
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The procedure of the xCELLigence RTCA-DP system (Roche) was previously described31 (link)70 (link). Briefly, 2 × 104 of each of serum-starved A549luc stable cell lines were suspended in 100 μl of serum-free medium, and then the cell suspension was added into the pre-equilibrated upper chamber of the CIM plate. The complete medium was added to the bottom-well of the plate, and a FBS-free medium was used as control. The plate was then inserted into the RTCA machine (housed within the incubator) and cell index values were detected every 15 min over the following procedure. The slopes of the curves at indicated time points were calculated using the RTCA software v1.2 (Roche Applied Science).
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