HCEC-1CT, SW620, and HT29 cells (2×103 cells in 200 μL medium/well) were seeded in E16-Plates (Acea Biosciences, Inc) according to the xCelligence Real Time Cell Analyzer (RTCA) DP manufacturer's instructions (Roche/Acea Biosciences). The growth was monitored with one sweep/hour for 145 hours in total. Analysis was performed using the RTCA 1.2.1.1002 software (Roche). The RTCA detects changes in the electrical impedance of gold electrodes on the well surface and thereby generates the so-called cell index. An increase in the cell index over time is a surrogate for active cell proliferation.
E plates 16
Manufactured by Agilent Technologies
Sourced in United States, Germany
The E-plates 16 are specialized lab equipment designed for cell-based assays. They provide a convenient platform for real-time monitoring and analysis of cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Xcelligence real time cell analyzer, by Agilent Technologies (2 mentions)
Xcelligence rtca sp, by Agilent Technologies (1 mentions)
Xcelligence dp system, by Agilent Technologies (1 mentions)
Charcoal stripped fbs, by Merck Group (1 mentions)
Xcelligence rtca dp system, by Agilent Technologies (1 mentions)
Rtca software v1, by Roche (1 mentions)
Xcelligence rtca dp analyzer, by Roche (1 mentions)
Prism software v5, by GraphPad (1 mentions)
Xcelligence system, by Roche (1 mentions)
Rtca sp instrument, by Roche (1 mentions)
Rtca software version 1, by Roche (1 mentions)
Hyclon, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
L glutamine, by PanEco (1 mentions)
Penicillin streptomycin, by PanEco (1 mentions)
Acy 1215, by Selleck Chemicals (1 mentions)
Xcelligence rtca dp, by Agilent Technologies (1 mentions)
Xcelligence system, by Agilent Technologies (1 mentions)
14 protocols using e plates 16
1
Real-Time Cell Proliferation Assay
2
Cell Proliferation Monitoring with xCELLigence
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Cells were seeded at a density of 0.5 × 105 and cultured for 3 days. Proliferation was monitored by means of an xCELLigence DP device (OLS OMNI Life Sciences, Bremen, Germany) for 72 h, using E-16 plates (#2801032, ACEA Biosciences Inc., San Diego, CA USA) following manufacturer’s guidelines.
3
Cell Adhesion Assays for CAFs and HCT8/E11
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Cell-to-cell adhesion assays between CAFs and HCT8/E11 cells were performed as described [39] . CT5.3hTERT cells were cultured in 24well plates until confluency. Subsequently, cells were incubated with solvent or Dex (1 µM) for 24 h prior to an additional seeding of 10 4 HCT8/E11-luc cells/well. After 24 h of co-culturing, cells were washed twice with DMEM in order to remove the non-adherent cells. Subsequently D-luciferine (150 μg/ml) was added to the wells and luciferase activity was measured using the IVIS system. HCT8/E11 cancer cells' adhesion to collagen coating was measured as described [30] . Briefly, HCT8/E11 cells (10 4 /well) were seeded in quadruplicates in type I collagen-coated (50 µg/ml) E-16 plates (ACEA Biosciences, Sand Diego, CA, USA). Cells were seeded in serum-free DMEM, CM CTRL and CM DEX . Cell-electrode impedance indicating cell adhesion was assessed every 5 min for 24 h using xCELLigence RTCA SP (ACEA Biosciences). Cell adhesion is reported as a relative cell index and areas under the curve (AUC) were calculated for the first 60 min of each treatment.
4
In vitro cytotoxicity evaluation of herbal-NLC
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xCELLigence technology and RTCA-DP analyzer have been employed for in vitro quantification of viability versus cytotoxicity of developed herbal-NLC. 1 × 104 HUVEC normal cells were seeded in 100 μL culture medium in each well (E-Plates 16, ACEA Biosciences, San Diego, CA, USA) and afterward were put in the xCELLigence DP-System and placed in a humidified incubator (5% CO2). The RTCA curves were automatically recorded on the xCELLigence System in real time. Cell impedance was measured (by using electronic sensors) and the small changes in impedance were continuously measured by RTCA-DP analyzer and after integration were expressed over time as Cell Index, CI (using RTCA Software 1.1, Mannheim, Germany). When cell proliferation reached a CI value over 1.0, scalar concentrations of NLC were added, and live cells were monitored.
5
Real-time Proliferation Monitoring of KLE Cells
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For real-time proliferation monitoring using the xCELLigence RTCA DP system (Agilent, Santa Clara, CA, USA), KLE cells were seeded onto E-plates 16 (ACEA Biosciences, San Diego, CA, USA) at cell densities of 5000 cells/well in growth medium without phenol red and with charcoal-stripped FBS (#F6765, Sigma-Aldrich). The next day, KLE cells were treated with different concentrations of CR extract (20–400 μg/mL; Table S2 ), only 50% ethanol, or only medium. The final concentration of ethanol was 2.5%. Experiments were repeated in two independent experiments, each time with four technical replicates for individual treatments. Cell proliferation was monitored for 180 h after treatment.
6
Real-Time Cell Proliferation Assay
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An xCELLigence Real-Time Cell Analyzer (ACEA Biosciences, San Diego, CA, USA) was used for analyzing HepG2 and Huh7 cell proliferation. Each well of an E-plates 16 (ACEA Biosciences) was filled with 3000 cells and monitored for 24 h. When the cells entered the log phase, 10 μL of STM was added in two independent experiments and monitored in the RTCA instrument for 72 h.
7
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.
8
Real-time Cell Proliferation Analysis in Lung Cancer
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Cell proliferation was analyzed in real-time using the commercially available impedance-based xCELLigence System (Roche Applied Science, Mannheim, Germany) in E-plates 16 (ACEA Biosciences, Inc) according to the manufacturer's instructions. The background signal of the culture medium was the first set up in an E-plate 16. Lung cancer cells were seeded in E-plates per the cell number obtained after titration in lung fibroblast cell-derived media (S4 Fig ); the cell numbers were 10,000 cells/well for NCI-H358, 40,000 cells/well for Calu-3, and 4,000 cells/well for A549. The signal was measured every 15 min, starting immediately after the seeding. Twenty-four hours after seeding, the culture media were removed and replaced with the asbestos-exposed lung fibroblast-derived media and asbestos-exposed lung cancer cell-derived media. Cell groups directly exposed to asbestos were also included. Normalized cell index (NCI) was calculated by dividing every CI at any given time by CI at the normalized point. The experiment was performed in triplicate or quadruplet.
9
Real-Time Monitoring of hBD-Mediated Cell Proliferation
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The xCELLigenceTM Real-Time Cellular Analysis (RTCA) system (Roche and ACEA Biosciences, California, USA) was used to monitor cell proliferation using impedance as the read out. The impedance measurement, displayed as cell index (CI) takes cell number, viability, and morphology into account; 1 × 104 HSC-1 or SCL-1 cells were treated with 20 µg/mL hBD-1, -2, and -3 (PeptaNova, Sandhausen, Germany) [13 (link)], respectively, and seeded in DMEM low glucose (1 g/L) with 1.5% FBS in E-plates 16 (ACEA Biosciences). Proliferation was monitored every 15 min by the xCELLigence system [76 (link)] for up to 24 h. Growth curves were normalized to the time point of cell adherence (~4 h). Evaluations were performed using xCELLigence 1.2.1 software (ACEA Biosciences). Each experiment was repeated three times.
10
Evaluating Endothelial Barrier Dynamics
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HUVEC were seeded to E-Plates®-16 (xCELLigence®, Acea Biosciences Inc.) at a density of 104 per well. Change of density of cells leads to a change of impedance, represented as cell index. To analyze a change of density after treatment normalized cell index was used. After pre-incubation in ECGM containing Supplement Mix and 10% FCS with 50 µM PETN or DMSO respectively for 24 h. To compare the formation of the barrier between control and PETN-treated cells cell index was then measured and compared. To analyze the effect of thrombin on endothelial barrier cells were treated with 10 U/ml thrombin in serum starved medium (ECGM without Supplement Mix or FCS) for up to 30 min. Barrier disruption leads to decreased cell index. The mean of the normalized cell index of duplicates was used for statistical analysis. Values were recorded over time as linear data.
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