Endothelial cell proliferation was measured by using an xCELLigence RTCA instrument (Roche Diagnostics) and E-plate 16 (a modified 16-well plate, Roche Diagnostics). The E-plate 16 was coated with 0.1% gelatin, loaded with 100 μL cell-free medium, and left in a tissue culture hood for 30 min to reach equilibrium. The E-plate 16 was placed into the RTCA instrument to measure the background impedance. Thereafter, 100 μL cell suspensions with fewer than 3500 cells were added into each well of the E-plate 16, which was then placed in a tissue culture incubator for 30 min to allow cells to settle down before being measured by the RTCA device. The impedance value of E-plate 16 was automatically monitored every 15 min. FGF2 was added at 100 ng/mL.
Xcelligence rtca instrument
Manufactured by Roche
Sourced in Germany
The XCELLigence RTCA instrument is a real-time cell analysis system designed to monitor cellular events in a label-free and continuous manner. The core function of the instrument is to measure cell adhesion, proliferation, and morphology changes over time.
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7 protocols using xcelligence rtca instrument
1
Endothelial Cell Proliferation Assay
2
Real-Time Cell Adhesion Dynamics
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The xCELLigence RTCA instrument (Roche) was used to measure cell adhesion over time. RTCA measures impedance between electrodes (bottom sensors) expressed as cell index values. The 96-well E-plate (Roche) was coated with 1 µg/ml fibronectin or 5 µg/ml vitronectin (A14700; Thermo Fisher Scientific) in PBS, followed by blocking with 0.1% BSA in PBS, both at 37°C for 1 h. The cells were detached and washed with full medium containing 10% FBS, and 10,000 cells/well were seeded on E-plates in serum-free medium. Cell index was measured in real time.
3
Real-time Cell Growth Monitoring
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Cell growth was monitored with a real-time x-Celligence RTCA instrument (Roche Applied Science, Penzberg, Germany) according to manufacturer’s instructions. 4000 cells per well were seeded in 200 μl media containing 10% FBS in an E-plate. The E-Plates were continuously monitored on a RTCA system for 72 h at 37 °C with 5% CO2. The impedance was measured as “cell index”. Data were collected and analyzed by RTCA software 1.2.
4
Real-Time Cell Adhesion Assay on Fibronectin
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The xCELLigence RTCA instrument (Roche) was used to measure cell adhesion on fibronectin in real-time (Hamidi et al., 2017 ). The RTCA instrument uses gold-bottom electrode plates to measure the impedance between two electrodes. This is expressed as an arbitrary cell index value. The xCELLigence 96-well plates (Acea Biosciences, E-Plate VIEW 96 PET, 00300600900) were coated with a solution of 20 μg/ml of poly-D-lysine (in PBS) for 1 h at 37°C, washed with PBS, and coated with a solution of 10 μg/ml fibronectin (in PBS) for 1 h at 37°C. Plates were then blocked using a solution of 1% BSA (in PBS) for 1 h in RT. After 2 PBS washes, 15000 cells were seeded into each well in a serum-free culture medium. The cell index was recorded over time.
5
Evaluating Radiation Sensitivity and Migration of U87 Glioblastoma Cells
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Control U87 or shLis-U87 cells were X-ray irradiated with doses from 5 to 50 Gy (RS2000 apparatus, Rad Source Technologies, Inc). Then, cells were seeded at 1x104 cells/ well, in quadruplicates or triplicates in E-plates and placed in xCelligence RTCA instrument (Roche), and followed-up for 100 hours. Alternatively, irradiated or non-irradiated cells were seeded in 24-well plates and cell proliferation assessed by DNA amount per well was determined using Hoechst 33342 (Molecular Probes), at day 2 and 9 post-irradiation.
To test cell adhesion and proliferation, CD133+ cells isolated from control U87 or from shLis-U87 were seeded on E-plates (104 cells/well), which were introduced in xCELLigence RTCA and followed-up for 3 hours (for cell adhesion tests) or 160 hours (for proliferation tests). To assess the capacity to migrate toward serum, CD133+ cells isolated from U87 or shLis-U87 were seeded at a density of 4x104 cells per well in the upper chamber of CIM plates; in the lower compartment, DMEM (control) or DMEM containing 20% FBS was added. The plate was placed in xCELLigence instrument and followed-up for 3 hours.
To test cell adhesion and proliferation, CD133+ cells isolated from control U87 or from shLis-U87 were seeded on E-plates (104 cells/well), which were introduced in xCELLigence RTCA and followed-up for 3 hours (for cell adhesion tests) or 160 hours (for proliferation tests). To assess the capacity to migrate toward serum, CD133+ cells isolated from U87 or shLis-U87 were seeded at a density of 4x104 cells per well in the upper chamber of CIM plates; in the lower compartment, DMEM (control) or DMEM containing 20% FBS was added. The plate was placed in xCELLigence instrument and followed-up for 3 hours.
6
Real-Time Cell Proliferation Monitoring
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The cell proliferation was continuously monitored by using the xCELLigence RTCA Instrument (xCELLigence RTCA, Roche, Germany) according to the manufacturer's instructions. CaCo-2 and Vero cells seeded at a density of 5 × 10 3 cells and 7 x 10 3 per well in 200 μl media containing 10% FBS in a 16-well E-plate (Roche Applied Science, Germany), respectively. After 24 h, drug-loaded sporopollenin of P. nigra, C. libani, and pure Oxaliplatin (control) were added at different concentrations (in the range of 5-20 mg/ml). The E-plates were continuously monitored on an RTCA system for 120 h at 37°C with 5% CO 2 . The proliferation of examined cells was monitored every 30 min and a time-dependent cell index (CI) graph was produced by the device using the RTCA software program of the manufacturer.
The baseline cell index for molecules-treated cells, compared to cancer and control cells, was calculated for at least two measurements from three independent experiments.
The baseline cell index for molecules-treated cells, compared to cancer and control cells, was calculated for at least two measurements from three independent experiments.
7
Cell Invasion and Gelatinase Zymography
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Cell invasion was also performed with the xCelligence RTCA instrument (Roche). In this assay, CIM-plate with two chambers were used. F-12 (180 µl) supplemented with 10% FBS was added to each well in the lower chamber. Cells were suspended in F-12 FBS-free media, then 60,000 cells/well were added to the uppper chamber. After attachment, cells invading through Matrigel (cat. no. 356234, BD Biosecience, China) towards lower chamber containing 10% FBS media were continuously monitored by the system and data were collected and analyzed by RTCA 1.2 software.
Gelatinase zymography. For the assessment of MMP-2 and -9, gelatin zymography assays were used. Gelatinase zymography was performed in an 8% SDS-PAGE gel in the presence of 0.1% gelatin under non-reducing conditions. Culture media with sample buffer were loaded for SDS-PAGE with Tris-glycine SDS buffer. Samples were not boiled before electrophoresis. Following electrophoresis, the gels were washed twice in 2.5% Triton X-100 for 30 min at room temperature to remove SDS. The gels were then incubated at 37˚C overnight in substrate buffer containing 50 mM Tris-HCl and 10 mM CaCl 2 at pH 8.0 and stained with 0.5% Coomassie Blue R250 in 50% methanol and 10% glacial acetic acid for 30 min and destained. Upon renaturation of the enzyme, the gelatinases digested the gelatin to produce clear bands against an intensely stained background.
Gelatinase zymography. For the assessment of MMP-2 and -9, gelatin zymography assays were used. Gelatinase zymography was performed in an 8% SDS-PAGE gel in the presence of 0.1% gelatin under non-reducing conditions. Culture media with sample buffer were loaded for SDS-PAGE with Tris-glycine SDS buffer. Samples were not boiled before electrophoresis. Following electrophoresis, the gels were washed twice in 2.5% Triton X-100 for 30 min at room temperature to remove SDS. The gels were then incubated at 37˚C overnight in substrate buffer containing 50 mM Tris-HCl and 10 mM CaCl 2 at pH 8.0 and stained with 0.5% Coomassie Blue R250 in 50% methanol and 10% glacial acetic acid for 30 min and destained. Upon renaturation of the enzyme, the gelatinases digested the gelatin to produce clear bands against an intensely stained background.
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