The invasive activity of PANC-1 cells was determined with xCELLigence® technology (ACEA Biosciences, San Diego, supplied by OLS, Bremen, Germany) as outlined in detail in previous publications [39 (link),40 (link)]. The lower side of the CIM plate-16 porous membrane was coated with a 1:1 mixture (v/v) of collagen I and collagen IV (30 μL) to facilitate adherence of the cells and thus enhance the duration of signal recording. Prior to cell seeding, the surface of the upper chamber was covered with a thin monolayer of 5% (v/v) growth factor-reduced Matrigel (BD Biosciences, Heidelberg, Germany) diluted 1:20 with basal medium, as detailed elsewhere [72 (link)]. After the Matrigel solification, each well was loaded with 60,000 or 80,000 cells in standard growth medium (see above). Cells were allowed to settle in the laminar flow hood for 30 min at RT, after which the assay was started and run for 24–48 h. Data acquisition (with signal recording every 15 min) and analysis was performed with the RTCA software (version 1.2, ACEA).
Xcelligence technology
Manufactured by Agilent Technologies
Sourced in United States
The XCELLigence technology from Agilent Technologies is a real-time cell analysis system that measures the electrical impedance of cells. This technology provides continuous, label-free monitoring of cell status, proliferation, and morphology changes in cell culture.
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Lab products found in correlation
E 16 well plates, by Agilent Technologies (3 mentions)
Rtca software, by Agilent Technologies (2 mentions)
Alamar blue fluorescent assay, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Sb431542, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Xcelligence rtca, by Agilent Technologies (1 mentions)
Igf 1, by Thermo Fisher Scientific (1 mentions)
Celltiter aqueous one solution cell proliferation assay, by Promega (1 mentions)
E plate, by Agilent Technologies (1 mentions)
18 protocols using xcelligence technology
1
Quantifying Invasive Potential of PANC-1 Cells
2
Real-Time Cell Proliferation Assay
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The xCELLigence technology (ACEA Biosciences Inc.) allows the Real Time Cell Analysis (RTCA) of cell proliferation without chemical interference. The RTCA instrument was used to assess the proliferation of different cell models overexpressing or invalidated for PK. All the experiments have been done after a titration assay that allowed us to determine the optimal cell seeding density for each cell line. After an automatic scan of the plate to check for proper contact of the E-plate, the background impedance for each well of the plate was measured before the cells were plated and incubated for 30 min at 37 °C and 5% CO2 to allow their settling in an evenly distributed pattern. The E-plate 16 was then put in the cradle pocket of the RTCA apparatus and the data acquisition was initiated. The impedance signals were recorded every 5 min for the first 25 scans (2 h) and every 15 min until the end of the experiment (up to 72 h).
In a subset of experiments, in order to determine the impact of secretates on the proliferation of murine glioblastoma GL261, cells (40,000 per well) were plated on an E-plate 16. Two-three hours after seeding, cells were treated without changing the media with extemporaneously produced secretates (50 µg of proteins) of TSM1 EV or PKWT cells. The proliferation was recorded as described above.
In a subset of experiments, in order to determine the impact of secretates on the proliferation of murine glioblastoma GL261, cells (40,000 per well) were plated on an E-plate 16. Two-three hours after seeding, cells were treated without changing the media with extemporaneously produced secretates (50 µg of proteins) of TSM1 EV or PKWT cells. The proliferation was recorded as described above.
3
Tryptase-Mediated Mast Cell Migration Assay
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To further investigate effect of tryptase on mast cell migration, a coculture system with HMC-1 and HMEC-1 cells was established. Migration of HMC-1 cells was assessed by using E-16-well plates and the xCELLigence technology (Acea Bioscience, San Diego, CA, USA) [17 (link)]. Briefly, 165 μL medium solutions containing tryptase at 0, 0.3, and 1.0 μg/mL with or without leupeptin (1.0 μg/mL) were added in lower chamber of E-16-well plates, respectively, and incubated for 1 h. For the upper chamber, 30 μL HMEC-1 cells (6 × 104 cells/well) were seeded in, and cells grew for 1 h. Anti-human ICAM-1 antibody (50 μL) was then added in specified wells and cultured for 15 min. This was followed by seeding 50 μL HMC-1 cell suspension (6 × 104 cells/well). All control wells received only the equal volume of medium. The HMC-1 migration number, expressed as a cell index value, was monitored for 48 h. Cells in the lower chamber were collected for flow cytometric analysis of HMC-1. The experiments were conducted in duplicate and repeated 4 times.
4
Cell Proliferation and Viability Assays
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Cell proliferation was assessed using E-16-well plates and xCELLigence technology (Acea Bioscience, San Diego, CA, USA, distributed by Roche).19 (link) Cell growth was monitored for 72 h. Microelectrodes, placed on the bottom of plates, were used to detect impedance changes proportional to the number of adherent cells and expressed as the cell index. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a cell index value. The experiments were conducted in triplicate and repeated twice.
For viability assays, melanoma cell lines were plated in 96-well plates at 5000 cells/well in complete medium. 24 hours after plating, varied doses of inhibitors were added in triplicate. 0.1% DMSO was used as negative control. Cell viability was evaluated after 72-hour incubation with drugs using Alamar Blue fluorescent assay (Life Technologies).
For viability assays, melanoma cell lines were plated in 96-well plates at 5000 cells/well in complete medium. 24 hours after plating, varied doses of inhibitors were added in triplicate. 0.1% DMSO was used as negative control. Cell viability was evaluated after 72-hour incubation with drugs using Alamar Blue fluorescent assay (Life Technologies).
5
Cell Proliferation Analysis via xCELLigence
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Cell proliferation was assessed using E-16-well plates and the xCELLigence technology (Acea Bioscience, San Diego, CA, USA, distributed by Roche Diagnostics) [35 (link)]. Briefly, cells (1 × 103 A375 cells/well) were seeded in E-16-well plates in complete medium and grown for 48 hours. Inhibitor drugs were then added as single agents or in different combinations (B-RAF inhibitor [GSK2118436] plus MEK inhibitor [GSK1120212], BRAF inhibitor plus AurkA inhibitor [MLN8054], MEK inhibitor plus AurkA inhibitor [MLN8054] or triple-combination of all three drugs) and the cell growth was monitored for an additional 72 hours. Microelectrodes, placed on the bottom of plates, were used to detect impedance changes proportional to the number of adherent cells and expressed as the cell index. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a cell index value. Doubling times for each cell line were calculated from the cell growth curve during the exponential phase. The experiments were conducted in triplicate and repeated twice.
6
Cell Proliferation Ability Assay
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Cell proliferation ability was performed using xCELLigence Technology (ACEA Bioscience, San Diego, CA, USA). A total of 4000 SV-HUC-1 cells/well were seeded in E-16-well plates (ACEA Bioscience, San Diego, CA, USA), and transfection was performed post 36 h of culturing the cells. IL17RA-NC-LPS group and IL17RA-OE-LPS group were treated with LPS (5 μg/mL) 36 h after transfection. Cell growth was monitored for another 24 h. Microelectrodes were placed at the bottom of the E-16 plate to detect changes in impedance proportional to the number of adherent cells. The impedance values for each well were automatically monitored by the xCELLigence system and expressed as cell index values.
7
Cell Proliferation and Migration Assays Using xCELLigence
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Cell proliferation was assessed using E-16-well plates and the xCELLigence technology (Acea Bioscience, San Diego, CA, USA, distributed by Roche Diagnostics) (43 (link)). Briefly, cells (4 × 103 OVCAR-3 cells/well) were transfected with non-target or target siRNA (Dharmacon) with RNAiMax (Invitrogen) using reverse transfection protocol in E-16-well. Cell growth was monitored for 72 h. Microelectrodes, placed on the bottom of plates, were used to detect impedance changes proportional to the number of adherent cells and expressed as the cell index. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a cell index value. The experiments were conducted in triplicate and repeated twice.
Cell migration assays were performed using an xCELLigence system (CIM-plates). Cell were transfected (1 × 104 in OPTI-MEM medium) with non-target or PAK1 siRNA and cultured for 72 hours. 105 cells were seeded in the upper chamber of the CIM-plates in serum-free media. The lower chamber was filled with RPMI-1640 medium containing 10% FBS. Cell index as acquired by the software was set to 100% migration after flattening of the slope. Experiments were performed in triplicate. The rate of cell migration was monitored in real-time with the xCELLigence system for duration of 48 h and expressed as a CI value.
Cell migration assays were performed using an xCELLigence system (CIM-plates). Cell were transfected (1 × 104 in OPTI-MEM medium) with non-target or PAK1 siRNA and cultured for 72 hours. 105 cells were seeded in the upper chamber of the CIM-plates in serum-free media. The lower chamber was filled with RPMI-1640 medium containing 10% FBS. Cell index as acquired by the software was set to 100% migration after flattening of the slope. Experiments were performed in triplicate. The rate of cell migration was monitored in real-time with the xCELLigence system for duration of 48 h and expressed as a CI value.
8
Cell Proliferation Assay using xCELLigence
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RBL-2H3/ETFR cell proliferation was assessed using E-16-well plates and the xCELLigence technology (Acea Bioscience, distributed by Roche Diagnostics) as described [12 (link)]. Briefly, cells (2x103/well) were seeded in 16-well plates in growth medium and left to growth for 90 hours in the presence or the absence of 10 μM [SRSRY] or diluents. Microelectrodes placed on the bottom of plates, detect impedance changes which are proportional to the number of adherent cells and are expressed as Cell Index. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value. Growth medium with/ without [SRSRY] was replaced every 24 hours. The experiments were performed at least twice in quadruplicate.
9
Cell Proliferation Assay using xCELLigence
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The proliferation analysis was performed using the xCELLigence technology (ACEA Biosciences, USA) according to the manufacturer’s instructions. A total of 20,000 cells were planted into each well of an ACEA E-plate 16 in triplicate. The cell index was registered every 10 min for 10–40 h. Results were represented as the mean ± SEM of three experiments.
10
Cell Proliferation Assay Using xCELLigence
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Cell proliferation was assessed using E-16-well plates and the xCELLigence technology (Acea Bioscience, distributed by Roche Diagnostics) as described [51 (link)]. Briefly, cells (2×103/well) were seeded in 16-well plates in growth medium and left to growth for 72 hrs. Microelectrodes placed on the bottom of plates, detect impedance changes which are proportional to the number of adherent cells and are expressed as Cell Index. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value. Doubling times for each cell clone were calculated from the cell growth curve during the exponential growth. The experiments were performed at least twice in quadruplicate.
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