Staphylococcal biofilm formation and removal was performed as previously described using the RTCA technology (Gutiérrez et al., 2016 (link); Gutierrez et al., 2017 (link)). Briefly, 100 μl of S. aureus 15,981 were diluted in TSBg (TSB supplemented with 0.25% w/v D-(+)-glucose) and poured into 16-well E-plates (∼106 CFU/well). E-plates were connected to the xCelligence RTCA-DP (ACEA Biosciences Inc., San Diego, CA, USA) holder which measure the impedance signal and represent cell index (CI) values. Biofilm were formed for 8 h at 37°C and then 100 μl of LysRODI and LysA72 were added to achieve a concentration gradient (0.14–9.15 μM and 0.21–13.42 μM, respectively) and incubated for an extra 16 h. Impedance values were further processed using the RTCA software 1.2.1 (ACEA Biosciences Inc.) as described previously (Gutierrez et al., 2017 (link)) in order to calculate: (i) the percentage of biofilm removal compared to control values after 16 h of treatment; (ii) the minimum biofilm eradicating concentration that removes 50% of the biofilm (MBEC50), (iii) the lowest antibiofilm effect (LOABE; lowest concentration needed to observe an antibiofilm effect) and (iv) the specific antibiofilm activity expressed as Δbaseline normalized CI × min−1 × mM−1. All the experiments were performed in triplicate.
Rtca software 1
Manufactured by Agilent Technologies
Sourced in United States
The RTCA Software 1.2 is a data analysis software developed by Agilent Technologies for use with their real-time cell analysis (RTCA) instrumentation. The software is designed to provide users with tools to monitor, analyze, and interpret data collected from RTCA experiments.
Automatically generated - may contain errors
Lab products found in correlation
16 well e plate, by Agilent Technologies (2 mentions)
Xcelligence rtca dp, by Agilent Technologies (1 mentions)
Xcelligence rtca sp system, by Agilent Technologies (1 mentions)
E plate, by Agilent Technologies (1 mentions)
8 well e plates, by Agilent Technologies (1 mentions)
Rtca icelligence system, by Agilent Technologies (1 mentions)
Rtca dp instrument, by Agilent Technologies (1 mentions)
Prism 9, by GraphPad (1 mentions)
Trypan blue dye, by Bio-Rad (1 mentions)
Xcelligence system, by Agilent Technologies (1 mentions)
Luna 2, by Logos Biosystems (1 mentions)
Xcelligence dp system, by Agilent Technologies (1 mentions)
Prism software, by GraphPad (1 mentions)
Xcelligence rtca dp instrument, by Agilent Technologies (1 mentions)
12 protocols using rtca software 1
1
Staphylococcal Biofilm Quantification and Removal
2
Real-time Monitoring of Cell Proliferation
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The real-time monitoring of proliferation was carried out with the xCELLigence RTCA SP system (ACEA Biosciences Inc.). Proliferative growth was monitored over 72 h by recording the impedance every 30 min. The data are presented as the CI (arbitrary units), which corresponds to changes in impedance. For the CI, slope and doubling time data were generated with RTCA Software 1.2.1 (ACEA Biosciences Inc.) as described by [62 (link)].
3
Intestinal Epithelium Model Using HT29 Cells
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The in vitro intestinal epithelium model was established using the HT29 (ECACC 91072201) cell line. The latter was purchased from the European Collection of Cell Cultures; cells were cultured in McCoy's 5a medium as described in del Rio, et al. (2017) . 2017). Briefly, stock solutions of spermine and spermidine were prepared in water and adjusted to pH 7. HT29 cells were seeded (2 x 10 4 cells/well) in 16-well E-Plates (ACEA Biosciences Inc.) and incubated for 20 h in a Heracell-240 Incubator (Thermo Electron LDD GmbH, Langenselbold, Germany) at 37°C under a 5% CO 2 atmosphere. The cell index was continuously monitored using RTCA software 1.2.1 (ACEA Biosciences Inc.). After incubation the cells were treated with different concentrations of spermine (0, 0.80, 1.20, 1.80, 2.40, 3.23, 3.50, 3.63, 4.01, 4.47, 4.49, 5.30, 6.00, 7.40, 9.80, 12.30 and 14.80 mM) or spermidine (0, 0.63, 1.25, 2.50, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 and 86 mM) . The final volume of culture medium (supplemented with either PA) in each well was 200 µl. The cell index was then monitored for another 24 h. For comparisons, the cell index was normalized to the time point just before the addition of the PA and set to 1. All experiments were conducted in at least triplicate under each set of each conditions.
4
Liver Fluke Granulin Impacts H69 Cell Proliferation
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Proliferation of H69 and ΔhuPGRN-H69 cells in response to exposure to 100 nM recombinant liver fluke granulin [17] , [21] was quantified using the impedance-based xCELLigence real time cell analysis (RTCA) approach (ACEA Biosciences). The liver fluke granulin peptide (∼10 kDa) was concentrated using Centripep with cut-off 3 kDa (Eppendorf) and resuspended in low salt solution, Opti-MEM. The absorbance at 205 nm and concentration of liver fluke granulin was determined by using a Nanodrop 2000c spectrophotometer (ThermoFisher) [31] (link). Five thousand cells/well were seeded in 16-well E-plates (ACEA) in H69 complete media. E-plates was inserted in the xCELLigence DP platform at 37 °C, 5% CO2 and changes in impedance reflecting cell adhesion and proliferation record at intervals of 20 min for 24 h. On the following day, the medium was removed and replaced with H69 complete medium supplemented with liver fluke granulin at 100 nM. Cellular proliferation was monitored for 48 h for the wild type H69, liver fluke granulin-treated H69, and ΔhuPGRN-H69 cells with and without liver fluke granulin treatment, and displayed here as change of impedance (Cell Index) after normalizing to the signals for the wild type H69, assigned as the reference cell line (RTCA Software 1.2, ACEA) [32] (link).
5
Effects of RNA Analogs on Engineered Cells
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The proliferation rate and survival of A549 human cells with shRNA-mediated PKR, RIG-I, and MDA5 knockdown as well as cells expressing scrambled shRNA (scr-shRNA), under snoRNA and snRNA analogs transfection were analyzed using the iCELLigence RTCA System (ACEA Bioscience, San Diego, CA, USA). Cells were plated in 8-well E-plates (ACEA Bioscience) at a density of 5000 cells per well in a total volume of 200 µL of IMDM, and were monitored in real-time mode (Figure S1 ). After the initial 24 h of growth, the culture medium was replaced with fresh IMDM with complexes of Lipofectamine RNAiMAX with snoRNA and snRNA analogs. The data was recorded every 15 min for 48 h post transfection, and cell indexes were calculated by RTCA Software 1.2 (ACEA Bioscience). Cell index is a parameter reflecting the impedance of electron flow caused by adherent cells. Relative cell indexes (RCI) for each ncRNA analogs were calculated as values relative to control incubated with Lipofectamine RNAiMAX only and normalized to effect on cells expressing scr-shRNA.
6
Real-Time Cell Proliferation and Migration
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The cell proliferation and migration were assessed by real-time monitor system with 16-well plates (E-plate 16 for proliferation and CIM-plate 16 for migration, ACEA BioSciences, Inc., San Diego, CA, USA) and the curves were monitored by an RTCA DP instrument (ACEA BioSciences, Inc., San Diego, CA, USA). For proliferation, cells were seededat 10,000 cells/well in FBS-containing medium. The plate was then monitored once every 30 s for 4 h and once every half hour thereafter. For evaluation of migration, 10% FBS RPMI medium was added to the lower chamber, then cells were seeded into the upper chamber at 20,000 cells/well with serum-free medium. The cell migratory activity was monitored every 10 s for 40 min and once every hour. The data were analyzed using RTCA software 1.2 (ACEA BioSciences, Inc., San Diego, CA, USA) [37 (link),38 (link)].
7
Multivariate Analysis of Cell Dynamics
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GraphPad Prism 9.4.1 was used for statistical analysis (GraphPad Software LLC, Boston/MA, USA). FACS data were analyzed using FlowJo 10.8.0 (FlowJo LLC, Ashland/OR, USA). xCELLigence RTCA data were analyzed using RTCA Software 1.2 (ACEA Biosciences).
8
Cell Viability and Proliferation Assay
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The viability and the number of cells were evaluated on the automated cell counter LUNA-II (Logos Biosystems, South Korea) using Trypan Blue Dye (Bio-Rad Laboratories). Cell proliferation was assessed by real-time cell analysis using electrical impedance assay—xCELLigence System (ACEA Bioscience, San Diego, CA, USA). RTCA software was used to determine CI values through the measured impedance recordings. Briefly, cells were plated in 16-well E-plates (ACEA Bioscience) at a density of 20,000 cells per well in a total volume of 200 µl of complete medium and were monitored in real-time mode. The data were recorded every hour for 62 h; cell indexes were calculated using RTCA Software 1.2 (ACEA Bioscience). Cell index is a parameter reflecting the impedance of electron flow caused by adherent cells.
9
Real-time monitoring of cell growth
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Cell growth was evaluated using the xCELLigence DP system (ACEA Biosciences, San Diego, CA), designed to monitor events in real time by measuring electrical impedance across interdigitated microelectrodes integrated on the bottom of tissue culture E-plates (ACEA), see http://www.aceabio.com/main.aspx [35 (link), 74 (link)]. H69 and HepG2 were seeded at 5,000 and 10,000 cells/well, respectively, into E-plates in medium supplemented with 10% fetal bovine serum (FBS), and cultured in a humidified incubator at 37°C, 5% CO2 for one day. The cells were rinsed in PBS and medium replaced with H69 or HepG2 medium diluted 1 in 20, i.e. 0.5% FBS, for four to six hours. Thereafter, the medium was replaced with conditioned medium and the cells monitored continuously, at intervals of 15 minutes, for up to 120 hours using the RTCA Software 1.2 (ACEA), as described [37 (link), 39 (link)].
10
Quantitative Data Analysis Protocols
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Flow cytometry data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA) and xCELLigence data were analyzed with RTCA Software 1.2 (ACEA Biosciences). Other analyses were performed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Statistical significance was accepted at p < 0.05 and is indicated by *(p < 0.05), **(p < 0.01), ***(p < 0.001), and ****(p < 0.0001).
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