Cancer cell migration was assessed using the Oris™ cell migration kit (Platypus), as previously described [8 (link)]. The (TCM-LEC)CM (100 μl) with or without 20 μM maraviroc (R&D Systems) or 200 μg/ml tocilizumab (Genentech) was added once the cancer cells had attached. Migration and proliferation assays using CIM (cell invasion and migration) plates and the RTCA system (ACEA Bioscience) were performed as previously described [9 (link)].
Rtca system
Manufactured by Agilent Technologies
Sourced in United States
The RTCA system is a real-time cell analysis platform that measures changes in cell status and behavior in a non-invasive manner. It provides quantitative and continuous monitoring of cell processes, such as cell proliferation, migration, and cytotoxicity, in a variety of cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Cim plate, by Roche (3 mentions)
E plate, by Roche (2 mentions)
Maraviroc, by R&D Systems (1 mentions)
Tocilizumab, by Genentech (1 mentions)
Anti il 8 antibody, by R&D Systems (1 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay kit, by Promega (1 mentions)
E plate 16, by Agilent Technologies (1 mentions)
24 well cell culture plate, by Corning (1 mentions)
Collagen r, by Serva Electrophoresis (1 mentions)
10 protocols using rtca system
1
Assessing Cancer Cell Migration
2
Real-Time Cell Growth Analysis
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Id1 KD HCT116 cells and respective controls were seeded onto cell culture E-plates (Corning; Corning, NY, USA) at a cell density of 1×105 (link) cells per well incubated in culture medium at 37 °C containing 5% CO2. The cell growth curves were automatically recorded on the RTCA system (ACEA Biosciences, Inc.; San Diego, CA, USA). The cell index was followed for 3 days.
3
Assessing Cancer Cell Migration
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Cancer cell migration was assessed by using the OrisTM Cell migration kit (Platypus Technology, Madison, WI), as previously described [38 (link)]. MDA-MB-231- fibroblast or macrophage conditioned media (100 μl) with or without 0.1 uM reparixin (MCE, Monmouth Junction, NJ) and anti-IL-8 antibodies (R&D Systems, 30 μg/ml) was added once the cancer cells had attached. Migration and proliferation assays using CIM-plates (Roche, Indianapolis, IN) and the RTCA system (ACEA Bioscience, San Diego, CA) were performed as previously described [47 (link)].
4
Assessing Chemotherapeutic Drug Cytotoxicity on Cancer Cell Lines
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Sensitivity of HepG2, A549 and SMMC-7721 cells to chemotherapeutic drug cytotoxicity was assessed by MTS as described previously (Pi et al, 2005 (link); Peng et al, 2016 ). CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay Kits were purchased from Promega (Madison, WI, USA). Cells were exposed to various concentrations of As2O3, 5-Fu, EPI or cisplatin with or without CPT at a determined concentration for 24 or 48 h. Measurements were expressed as percentage change from untreated control (Vehicle) of appropriate cells. The lethal concentration 50 (LC50) values were determined from analysis of the log-linear phase of the curves.
The cell growth and proliferation of HepG2 cells were determined using the RTCA system (ACEA Biosciences, San Diego, CA, USA). Cell culture media (50 μl) were placed in each well of the E-plate 16 (ACEA Biosciences, San Diego, CA, USA). The E-plate 16 was then connected to the RTCA system to obtain background impedance readings. A total of 20 000 cells in 100 μl were incubated in the RTCA system overnight. Cells were treated with 10 μM As2O3, 200 μM 5-FU or 4 μM EPI with or without 0.1 μM CPT for 24 h on the RTCA SP Station located in an incubator at 37 °C with 5% CO2. Cell index values for cell activities were measured by continuous impedance recording every 5 min (Ren et al, 2011 (link)).
The cell growth and proliferation of HepG2 cells were determined using the RTCA system (ACEA Biosciences, San Diego, CA, USA). Cell culture media (50 μl) were placed in each well of the E-plate 16 (ACEA Biosciences, San Diego, CA, USA). The E-plate 16 was then connected to the RTCA system to obtain background impedance readings. A total of 20 000 cells in 100 μl were incubated in the RTCA system overnight. Cells were treated with 10 μ
5
Quantifying HUVEC Migration and Adhesion
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HUVEC migration was assessed, using CIM-plates (Roche) and the RTCA system (ACEA Bioscience); adhesion was assessed using E-plate (Roche) in the RTCA system55 (link). Briefly, the membrane of the top chamber of a CIM-plate was coated with fibronectin by adding 40 µL of 20 µg mL−1 fibronectin dissolved in PBS and incubating at 37°C for 30 min. 180 µL of EGM-2 (complete media for HUVECs) or EBM (serum free media) or MB231-LEC CM was added to the bottom chambers. The equilibrated plate was removed from the incubator and 100 µL of the trypsinized cells (45,000 HUVECs per well) with or without inhibitors were added to the top chamber. After 30 min incubation at room temperature, the stabilized chamber was loaded in the RTCA machine and the cell index was measured continuously for 20 h. Cell indices at 20 h were selected for analysis. ACEA E-plates (Roche Diagnostics) were used to measure the extent of HUVEC adhesion. Briefly, HUVECs (25,000 cells per well) in 100 µL of EGM-2 (complete media for HUVECs) or EBM (serum free media) or MB231-LEC CM were added. After equilibrating at room temperature for 30 min, the E-plate was loaded into the RTCA personal system. Cell indices at 3 h were analyzed.
6
Quantifying HUVEC Migration and Adhesion
7
Real-Time Cell Viability Monitoring
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According to previous reports (Stoddart 2011 (link); Fabio Cerignoli et al., 2018 (link)), the RTCA system (ACEA Biosciences, DP) continuously measured cell viability in real-time through the impedance readout. The arbitrary unit reflecting the electronic cell-sensor impedance is called the cell index (CI). We added 50 μl of ASC culture medium to the wells of 16-E-Plates (ACEA Biosciences; n = 4) and measured the background impedance. The vector-GFP- and circ-ATXN2–GFP-expressing cells were seeded at a density of 2,000 cells per well of a 16-E-Plate in 100 μl of culture medium_ and allowed to passively adhere to the electrode surface. The 16-E-Plate was placed inside a laminar flow hood for half an hour at 24–26°C before being transferred into a cell culture incubator with an RTCA instrument. Data were recorded at 15-min intervals throughout the entire experiment once the plates were properly placed.
8
LPS-Induced Injury in HPAEC Cells
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HPAEC cells between P5 to P8 were inoculated into a 16-well E-Plate (ACEA Biosciences, San Diego, CA, USA) at a concentration of 2 × 104 per well in 100 µl, then cultured in an incubator at 37 °C and 5% CO2 for 12 h following which LPS was added at varying concentrations of 0, 0.5, 1, 2 and 5 µM. The 16-well E-Plates were placed within the Real Time Cellular Analysis (RTCA) System (ACEA Biosciences) and cultured in an incubator at 37 °C and 5% CO2. Micro-electrical impedance of HPAEC cells were detected in real-time to investigate the effects of acute injury caused by LPS on HPAEC cells, at different concentrations.
HPAEC cells between P5 to P8 were inoculated into a 24-well cell culture plate (Corning, Inc., Corning, NY, USA) at a concentration of 1 × 105 per well in 600 µl, then cultured in incubator at 37 °C and 5% CO2 for 6 to 12 h after LPS was added at a final concentration of 1 µM. Cells were collected at different time points and the expression of TNF-α was detected to investigate the acute injury effects of LPS on HPAEC cells.
HPAEC cells between P5 to P8 were inoculated into a 24-well cell culture plate (Corning, Inc., Corning, NY, USA) at a concentration of 1 × 105 per well in 600 µl, then cultured in incubator at 37 °C and 5% CO2 for 6 to 12 h after LPS was added at a final concentration of 1 µM. Cells were collected at different time points and the expression of TNF-α was detected to investigate the acute injury effects of LPS on HPAEC cells.
9
Automated Real-Time Cell Migration Assay
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We used a real-time cell analyzer (RTCA) system (ACEA, USA) to explore the effect of PFN2 on endothelial cell migration and proliferation, as described in our previous report (27 (link)). Briefly, cells were seeded in E-plates at a density of 1,000 HUVECs/well and 2,000 RBMECs/well. The E-plates were then transferred to the RTCA-Dual Purpose instrument for automated real-time monitoring under standard incubator conditions. Cell index measurements were collected every 5 min. Cellular migration and invasion were also monitored using the RTCA system on cell invasion-and-migration (CIM)-plates instead of E-plates. Cell migration activity was monitored with the impedance readouts. Migration assays were performed by seeding cells in the upper chambers of the CIM-plates in serum-free medium at a density of 10,000 cells/well. The bottom chambers of the CIM-plates were filled with serum-containing medium to promote migration across the membranes along the serum gradient. Data were collected by real-time readouts.
10
Evaluating Hepatocyte-CD8 T Cell Interactions
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The xCELLigence system (Real-Time Cell Analysis (RTCA) System, ACEA Biosciences Inc., San Diego, CA, USA) was used to determine the cell-viability of target cells during co-culture experiments. Primary murine hepatocytes were isolated from non-infected C57Bl/6 mice as described previously [32 (link)]. A total of 1 × 104 primary murine hepatocytes were seeded per well of a E-96-wellplate coated with 0.02%, collagen R (#47254.02, SERVA Electrophoresis GmbH, Heidelberg, Germany). Then, 24 h after attachment, hepatocytes were infected with Ad-HBV1.3 (MOI 5) or left untreated and then cultured for a further 48 h. Thereafter, primary murine hepatocytes were co-cultured with 5 × 105 CD8 T cells from the livers of mice infected either 107 pfu or 108 pfu Ad-HBV-Luc. Intrahepatic CD8 T cells were isolated by Percoll gradient followed by magnetic bead separation (#130-096-543, Miltenyi Biotec, Bergisch Gladbach, Germany). Cell viability is illustrated as the cell index normalized to the start of co-culture.
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