Huh7 cells were transfected in 6-well plates and after 72-h incubation, 1250 cells/well were seeded into E-plate 96. Cell proliferation was monitored every 30 min for 96 h using xCELLigence system (Roche Applied Science) that allows performing real-time and label-free cellular analyses. Changes in proliferation were expressed as cell index which is defined as (Rn−Rb)/15, where Rb is the background impedance and Rn is the impedance of the well with cells. Doubling time was determined with RTCA software 1.2.1 (Roche Applied Science). The time required to reach a cell index value of 4 from 2 was calculated for each group.
Rtca software 1
Manufactured by Roche
Sourced in France, Germany, Switzerland
RTCA Software 1.2 is a software application developed by Roche to support the functionality of Roche's real-time cell analysis (RTCA) instruments. The software is used to monitor and analyze cellular behavior in real-time, providing researchers with data on cell proliferation, migration, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Xcelligence system, by Roche (3 mentions)
Xcelligence rtca dp system, by Roche (1 mentions)
Matrigel, by Corning (1 mentions)
E plate 16, by Roche (1 mentions)
Xcelligence rtca sp system, by Roche (1 mentions)
E plate view 96, by Roche (1 mentions)
Xcelligence mp, by Roche (1 mentions)
Xcelligence rtca mp instrument, by Roche (1 mentions)
Ls 7500, by Beckman Coulter (1 mentions)
Methyl 3h thymidine 50 ci mmol, by Cytiva (1 mentions)
Prism 6, by GraphPad (1 mentions)
Xcelligence system, by Agilent Technologies (1 mentions)
Icelligence system, by Agilent Technologies (1 mentions)
E plate, by Roche (1 mentions)
E plates 16, by Roche (1 mentions)
Rtca dp instrument, by Roche (1 mentions)
Lionheart fx, by Agilent Technologies (1 mentions)
Gen5 3, by Agilent Technologies (1 mentions)
F 2500, by Hitachi (1 mentions)
Prism 7, by GraphPad (1 mentions)
23 protocols using rtca software 1
1
Real-Time Cell Proliferation Assay
2
Real-Time Monitoring of Cell Invasion
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Cell invasion was examined in real time using the xCELLigence RTCA DP System (Roche Applied Science, Penzberg, Germany). The xCELLigence system (Roche Applied Science) allows continuous quantitative monitoring of cellular behaviour including invasion by measuring electrical impedance at a porous membrane (pore size 8 μm). U87 cells were seeded at 22 500 cells/well into specialized two‐layer cell invasion and migration plates coated with 20 μL matrigel (Corning, 1 : 30 dilution) and cultured without FBS for 24 h in the presence or absence of Q‐PAC. Lower layer wells were filled with Dulbecco's modified Eagle's medium/F12 with 10% FBS as chemoattractant. Invasion was continuously monitored in real time over a period of 24 h. Data analysis was carried out using RTCA Software 1.2.1 (Roche Applied Science) supplied with the instrument.
3
Real-Time Cell Growth Monitoring
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The cell index was monitored with an xCELLigence Real-Time Cell Analyzer (RTCA) instrument with E-Plate 16 (Roche) according to the manufacturer’s protocol. The cell index was normalized with the RTCA software 1.2.1 (Roche).
4
Dynamic Proliferation Assay of NC Cells
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NC cells were seeded in E-96-well plates and infected 24 h later with T-VEC. At 1 hpi distinct SMIs were added. Real-time dynamic cell proliferation was measured every 30 min during the full observation time of 96 h by xCELLigence RTCA SP System (Roche Applied Science, Penzberg, Germany). The cell impedance, i.e., the electrical resistance to alternating current, was expressed in an unspecified unit as the cell index. Cell index values were calculated using the RTCA software (1.2.1; Roche Applied Science, Penzberg, Germany).
5
Real-time cell cytotoxicity assay
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For impedance measurements we used the xCELLigence System (Roche) that provides a highly sensitive method to measure the cell index as a proxy of cell adhesion, proliferation, cell death and morphological alterations. Cell lines were subconfluent, and each cell line has been optimized in previous titration experiments so that cell type-specific optimal conditions for the seeding rate and confluence were obtained for the real-time measurement in these experiments. Cells were seeded at 5000 cells per well in quadruplicates in E-Plate VIEW 96 (Roche) and left untreated for 24 hrs within the RTCA SP station positioned in a 37°C incubator with 5% CO2 supply. For monitoring compound mediated cytotoxicity, cells were then treated with DMSO only or with 0.2 μM, 1 μM or 5 μM olaparib and incubated for an additional 72 hrs. The Cell Index was plotted and analysed using RTCA software 1.2.1 (Roche).
6
Real-Time Impedance Monitoring of Cell Proliferation
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Real-time label-free impedance-based monitoring of cellular proliferation assay was performed by using xCELLigence MP (Roche Applied Science). Transfected cells were incubated in 6-well plates for 48 h. After the incubation period, 5000 cells/well were seeded in E-plate 96. Cell proliferation was monitored at every 15 min for 72 h. Changes in proliferation rate were expressed as “cell index” (RTCA software 1.2.1, Roche Applied Science).
7
Real-Time Chemosensitivity Monitoring of Liposarcoma
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For chemosensitivity analysis, the SW872 cells were incubated in co-culture with TAFs or NFs for 48 h. Thereafter, the SW872 cells were seeded in two 8-well plates with an integrated microelectronic sensor array (iCELLigence Real-Time cell analyzer; CEA Biosciences, San Diego, USA). After 24 h, the cultivated human liposarcoma cells were incubated with doxorubicin at a concentration of 0.25 µg/ml that exhibited strong pro-apoptotic effects in previous a in vitro study (42 (link)). Cell proliferation and survival were monitored real-time by measuring cell-to-electrode responses of the seeded cells. The cell index (CI) was calculated for each E-plate well using RTCA software 1.2 (Roche Diagnostics, Meylan, France), as previously described (43 (link)). CI was calculated for each E-plate well using RTCA Software. The graphs are real-time generated outputs from the iCELLigence system.
8
Cell Migration Assay Using xCELLigence
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For the migration assay the cells were transfected as described above. Cells were cultured in serum free medium for 24h. After the starving 5 × l04 HEC1B cells in 100 μ1 were seeded in a CIM 16 well E-plate. The continuous migration of the cells was documented and analysed in a time-resolved manner with the xCELLigence RTCA MP instrument and the RTCA Software 1.2 (Roche diagnostics, Mannheim, Germany) to measure the CI values. The migration was measured over a time period of 48h. During the measurement the lower chambers contained full growth medium as chemoattractant and the upper chambers medium without FCS.
9
Measuring Human Mesangial Cell Proliferation
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[3H]-thymidine incorporation was measured to determine the effect on human mesangial cell proliferation. The mesangial cells were plated at 2.5 × 104 cells/well in 24-well cell culture plates for 24 h, and the medium was replaced with serum-free medium containing 0.1% bovine serum albumin and the incubation continued for another 24 h. Quiescent cells were treated with 10 μM angiotensin II and DS-EA, respectively, and 1 µCi of [3H]-thymidine was added (methyl-[3H] thymidine 50 Ci/mmol; Amersham, Oakville, ON, Canada). After incubation for 24 h, cells were extracted three times with cold 10% TCA for 5 min each time and solubilized for at least 30 min in 0.3 N NaOH. After neutralizing, [3H]-thymidine activity was measured in a liquid scintillation counter (Beckman LS 7500, Fullerton, CA, USA). Each experiment was performed in triplicate or quadruplicate. Mesangial cell index was calculated for each E-plate well by RTCA Software 1.2 (Roche Diagnosis, Meylan, France). The graphs are real-time generated outputs from the iCELLigence system.
10
Cellular Ligand Response Analysis
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Data was analyzed as stipulated in the previous protocol [21] . Briefly, experimental data was obtained with RTCA Software 1.2 (Roche Applied Science). Ligand responses were normalized to Δ cell index (Δ CI) and exported to GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) for further analysis. Vehicle control was subtracted as baseline to correct for any agonist-independent effects. Peak responses were defined as highest Δ CI (Max ∆CI) observed within 60 minutes after compound addition. When stipulated, area under the curve (AUC ∆CI) within those 60 minutes was used as an additional parameter to analyze response height. Peak values and experimental Δ CI traces were used for construction of bar graphs or concentrationeffect curves by nonlinear regression and calculation of IC50, EC50 and EC80 values. KI values for antagonists were calculated using the Cheng-Prusoff equation [34] using the concentration of the agonist (CGS21680, 100 nM) and EC50 value corresponding to each cell line.
All values obtained are means of at least three independent experiments performed in duplicate, unless stated otherwise. Statistical significance was determined by comparison of the means of multiple data sets by one-way ANOVA, followed by a Tukey's post-hoc test for comparison of all columns or a Dunnett's post-hoc test when comparing to control or reference compound.
All values obtained are means of at least three independent experiments performed in duplicate, unless stated otherwise. Statistical significance was determined by comparison of the means of multiple data sets by one-way ANOVA, followed by a Tukey's post-hoc test for comparison of all columns or a Dunnett's post-hoc test when comparing to control or reference compound.
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