VEC proliferation was assessed using a cellular impedance assay. VECs were seeded into 8-well E-plates (ACEA Biosciences, Inc., San Diego, CA, USA) at a density of 7,500 cells/well and cultured overnight. Cells were cultured with complete medium, supplemented with 0.1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich; Merck KGaA) as a control, the PPARγ agonist rosiglitazone (10 µM; Sigma-Aldrich; Merck KGaA) or the PPARγ antagonist T0070907 (15 µM; Sigma-Aldrich; Merck KGaA) respectively, as previously described (28 (link),29 (link)). Cellular proliferation was dynamically monitored using the iCELLigence™ real-time cell analysis (RTCA) system (ACEA Biosciences, Inc.) at 37°C in a 5% CO2 atmosphere for 100 h. The cell index, which reflects the adhesion, proliferation and viability of the cells through electrical impedance across interdigitated microelectrodes integrated on the bottom of the E-plates, was automatically calculated for each E-plate well using RTCA software version 1.2 (Roche Diagnostics, Basel, Switzerland) and graphs were generated in real-time using the iCELLigence™ system (30 (link),31 (link)). Each treatment was performed in duplicate and three independent experiments were conducted.
8 well e plates
Manufactured by Agilent Technologies
Sourced in United States
The 8-well E-plates are a lab equipment product designed for cell-based assays. They provide a platform for real-time monitoring of cellular events.
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Lab products found in correlation
3 protocols using 8 well e plates
1
Real-Time Monitoring of VEC Proliferation
2
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.
3
Real-Time Cell Impedance-Based Assay
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The real-time cell electronic sensing assay is based on electrical impedance readings in cell monolayers plated in wells containing built-in gold electrodes. ACEA RT-CES analyzer, 8-well e-plates, and the integrated software which we have used were from Acea Biosciences Inc (San Diego, CA). Cells were plated at a density of 1 × 104 cells/well in 250 μL of medium. The analyzer and the installed plates were placed in a standard cell culture incubator, at 37°C in a humidified atmosphere of 5% CO2. Cells were allowed to adhere to plates overnight. After cells seeded, the analyzer was programmed to take readings during 0-96 h, and WZ35 at 0.4, 2.0 or 10 μM was added to the medium at 28 h after incubation. Data were recorded and analyzed using the integrated software. The cell index is a quantitative measure of the spreading and/or proliferative status of the cells in an electrode-containing well.
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