Impedance was measured at 10 kHz by an RTCA SP instrument (ACEA Biosciences, San Diego, CA, USA). This method is label-free, non-invasive, and monitors cell adherence, growth, and viability real time. We have successfully tested the cellular effects of pharmaceutical excipients and peptides by impedance kinetics in our previous studies.32–34 (link) For background measurements 50 μL of cell culture medium was added to the wells, then cells were seeded at a density of 5×103 cells/well to 96-well plate with gold electrodes (E-plate 96, ACEA Biosciences) coated with collagen. Cells were cultured for 4–5 days in a CO2 incubator at 37°C and monitored every 10 minutes until the end of experiments. Cells were treated at the beginning of the plateau phase of growth. The treatment solutions were dissolved in Ringer buffer. Triton X-100 (TX-100) detergent (1 mg/mL) was used as a reference compound to induce cell toxicity. Cell index was defined as Rn-Rb at each time point of measurement, where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the background impedance of the well with the medium alone.
Rtca sp
Manufactured by Agilent Technologies
Sourced in United States
The RTCA SP is a real-time cell analysis system from Agilent Technologies. It measures the dynamic changes in electrical impedance of cells growing on specialized microelectronic plates. This provides quantitative and label-free data on various cellular parameters such as cell viability, proliferation, and cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Pet 16 e plate, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium glutamax, by Thermo Fisher Scientific (2 mentions)
Xcelligence system, by Agilent Technologies (2 mentions)
E plate 96, by Agilent Technologies (2 mentions)
E plate, by Agilent Technologies (2 mentions)
96 well e plate, by Agilent Technologies (1 mentions)
10 protocols using rtca sp
1
Real-Time Cellular Impedance Assay
2
Real-Time Cell Proliferation Monitoring
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The xCELLigence system (RTCA-SP, ACEA Biosciences Inc.) was used according to the manufacturer’s instructions for the continuous real-time monitoring of cell adhesion and proliferation by cell-electrode impedance57 (link) displayed as the dimensionless Cell Index (CI). By using an RTCA Analyzer, electrical impedance changes were measured across interdigitated microelectrodes integrated on the bottom of a specialized 96-well plate (E-Plate 96) and sent to the RTCA control subunit. The latter used RTCA Software (version 2.0) for CI calculations from the frequency-dependent electrode resistances and real-time display of data.
The background impedance of E-Plate 96 wells was determined growth medium only. Subsequently, 5,000 cells/well were plated in growth medium. A relatively low cell number was used to enable exponential growth and examination of the proliferation capacity. Then, localized on the RTCA SP Station, the E-Plate 96 was placed into the CO2-incubator, and the CI was monitored every 15 minutes over a period of 90 hours. RTCA Software was used to calculate the doubling time (DT).
The background impedance of E-Plate 96 wells was determined growth medium only. Subsequently, 5,000 cells/well were plated in growth medium. A relatively low cell number was used to enable exponential growth and examination of the proliferation capacity. Then, localized on the RTCA SP Station, the E-Plate 96 was placed into the CO2-incubator, and the CI was monitored every 15 minutes over a period of 90 hours. RTCA Software was used to calculate the doubling time (DT).
3
Quantitative Adhesion and Proliferation Assay
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In order to compare the functionality of MPC/SATC isolated from male DUC and DUhTP mice, growth kinetics, adhesion, and proliferation properties were continuously recorded over 72 h using the xCELLigence system (RTCA-SP, ACEA Biosciences Inc., San Diego, CA, USA), which measures impedance changes caused by the attachment and spreading of cells and calculates them as the dimensionless Cell Index (CI).
Therefore, forty thousand cells per well in quadruplicates were used in a 96-well-plate to assess quantitative adhesion and proliferation capabilities. The parameters slope (expressing the adhesion kinetics; AP), maximal cell index (highest CI value, usually associated with the stationary growth phase, here: end point value of the CI; CImax), and doubling time (DT, a measure of proliferation rate) were used and are shown exemplarily inFigure 2 [46 (link),47 (link)].
More details on the xCELLigence technology can be found in Atienza et al. (2005) or Ke et al. (2011).
Therefore, forty thousand cells per well in quadruplicates were used in a 96-well-plate to assess quantitative adhesion and proliferation capabilities. The parameters slope (expressing the adhesion kinetics; AP), maximal cell index (highest CI value, usually associated with the stationary growth phase, here: end point value of the CI; CImax), and doubling time (DT, a measure of proliferation rate) were used and are shown exemplarily in
More details on the xCELLigence technology can be found in Atienza et al. (2005) or Ke et al. (2011).
4
Real-time Analysis of H9C2 Cell Responses to UK5099
5
Real-time Analysis of H9C2 Cell Responses to UK5099
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The xCELLigence system (Acea Biosciences, RTCA SP) was used for real-time and time-dependent analysis of H9C2 cell responses to UK5099 (10 μM or 20 μM). 20,000 cells were seeded on an PET 16 E-Plate and cultured in 250 μl of Dulbecco’s Modified Eagle Glutamax Medium (Thermo Fisher Scientific). After 24 hours, cells were treated with vehicle or UK5099 and real-time cell index was continuously measured for the following 96 hours using the cell impedance detection system. Cell Index is a measure of impedance which correlates with the number, attachment and size of the cells.
6
Impedance-Based Monitoring of Niosome-RBEC Interactions
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Kinetics of primary RBEC responses to niosomes were monitored by impedance measurement (RTCA-SP; Agilent Technologies, Santa Clara, CA, USA), which is label-free, real-time, non-invasive and correlates linearly with the viability of cells [20 (link),21 (link)]. After background measurements with culture medium, RBECs were seeded at a density of 6 × 103 cells/well in 96-well plates with integrated gold electrodes (E-plate 96, Agilent), coated with collagen type IV (100 µg/mL) and fibronectin (25 µg/mL). When the impedance of cells reached a plateau phase indicating a confluent culture, cells were treated with niosomes (0.3, 1 or 3 mg/mL), and their impedance was monitored every 5 min for 24 h. Cell index was defined as Rn-Rb at each time point of measurement, where Rn is the impedance of the well when it contains adherent cells, and Rb is the background impedance of the well containing culture medium alone. Cell index was normalized in each well to the value measured at the last timepoint before the treatment.
7
Real-time Impedance Assay for BEC Viability
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Kinetics of the viability of BECs was observed by a real-time impedance measurement (RTCA-SP, Agilent, Santa Clara, CA, USA) as described previously [37 (link)]. Impedance correlates linearly with cell number, adherence, growth and viability. BECs were grown on golden electrodes of 96-well E-plates (Agilent) in a CO2 incubator at 37 °C for 5 days. Then the cells were treated with cytokines at 10 or 50 ng/ml concentration in 3 combinations: IL-6, IL-10 and IL-6 + IL-10. Effects of treatments were monitored for 24 h, as shown for IL-6 in Additional file 1 : Fig. S1.
8
Viability Kinetics of Brain Endothelial Cells
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Kinetics of the viability of brain endothelial cells after fasudil treatment was monitored by real-time impedance measurement (RTCA-SP, Agilent, Santa Clara, CA, USA). Impedance measurement correlates linearly with cell number, adherence, growth, and viability [30 (link)]. The RTCA-SP system (Agilent, Santa Clara, CA, USA) registers the impedance of cells automatically every 10 min. Impedance (SI unit: Ω, ohm) is expressed as an arbitrary unit called cell index. For every time point cell index is defined as (Rn − Rb)/15, where Rn is the impedance of the wells containing cells and Rb means the background impedance of the wells containing medium but not cells. RBEC were seeded at a cell number of 5 × 103/well onto a 96-well E-plate (Agilent) with golden electrodes at the bottom of the wells, and were kept in the CO2 incubator at 37 °C for 4–5 days. Cells were treated at the beginning of the plateau phase of cell growth with 0, 0.1, 0.3, 1, 3, 10, 30 and 100 μM concentrations of fasudil. Effects of the treatment were followed for 24 h.
9
Real-Time Analysis of Brain Endothelial Cells
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Using a label-free, noninvasive, impedance based technique (RTCA SP, Agilent, Santa Clara, CA, USA) confluency, morphology and barrier integrity of primary rat brain endothelial cells can be monitored in real time. This technique correlates linearly with the cell number, adherence, growth and viability of the cells [27 (link), 33 (link)]. This method is also a good verification for end-point colorimetric tests, such as MTT assay [33 (link)]. Here primary rat brain endothelial cells at a cell number of 5 × 104 cells/well were seeded on special 96-well plates with embedded gold electrodes (E-plate, Agilent). First, background impedance was measured with the addition of 50 µl cell culture medium, then cells were added and growth was followed. Once cells reached the plateau phase of growth they were treated with 1–40 mM l -ornithine or d -ornithine, or with the reference compound 1% Triton-X100 detergent to induce cell death. Effects of the treatment was followed for 24 h.
10
Fasudil's Effect on Brain Endothelial Cells
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Kinetics of the viability of brain endothelial cells after fasudil treatment was monitored by real-time impedance measurement (RTCA-SP, Agilent, Santa Clara, CA, USA). Impedance measurement correlates linearly with cell number, adherence, growth, and viability [29] . RBEC were seeded at a cell number of 5 × 10 3 /well onto a 96-well E-plate (Agilent) with golden electrodes at the bottom of the wells, and were kept in the CO 2 incubator at 37°C for 4-5 days. Cells were treated at the beginning of the plateau phase of cell growth with 0, 0.1, 0.3, 1, 3, 10, 30 and 100 µM concentrations of fasudil. Effects of the treatment were followed for 24 h.
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