The xCELLigence Real-Time Cell Analyzer (RTCA) system (Acea Biosciences, San Diego, CA, USA) was adopted to measure cell damage induced by Candida pathogens. VK2/E6E7 cells were counted and then seeded (5×104) into the E-plate 96 wells, incubating at 37°C for at least 20 h prior to treatment. When the cell growth curve reached a plateau, inhibitors or DMSO (0.8%) were added to the host cells 2h prior to the fungal stimulation and the vaginal epithelial cells were infected by different Candida strains at MOI of 10. EGFR inhibitor AG1478(Absin, abs810610) was used at 4.5μM, ERK1/2 inhibitor SD5978(Biyotime) was used at 5μM and p38 inhibitor SB203580 (selleckchem) was used at 5uM. The cell index was automatically recorded every 15 min during the incubation period. This experiment was performed in duplicates and run for 24h postinfection.
Xcelligence real time cell analyzer system
Manufactured by Agilent Technologies
Sourced in United States
The XCELLigence Real-Time Cell Analyzer System is a laboratory instrument used for monitoring and analyzing cell behavior in real-time. It provides continuous, label-free, and non-invasive measurements of various cellular parameters such as cell proliferation, cell viability, and cell-substrate interactions.
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Lab products found in correlation
Real time cell analyzer integrated software, by Agilent Technologies (2 mentions)
Rtca integrated software, by Agilent Technologies (2 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Columbus system, by PerkinElmer (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Xf96 extracellular flux analyser, by Agilent Technologies (1 mentions)
Luciferin substrate, by GoldBio (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Incucyte s3 live cell analysis system, by Sartorius (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Matrigel invasion chamber, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Anacardic acid, by Merck Group (1 mentions)
Trichostatin a, by Merck Group (1 mentions)
Cl amidine, by Cayman Chemical (1 mentions)
Sf1670, by Merck Group (1 mentions)
Cytometric bead array, by BD (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
26 protocols using xcelligence real time cell analyzer system
1
Real-Time Monitoring of Candida-Induced Cell Damage
2
Real-time Cytotoxicity Assay of CAR-T Cells
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The cytotoxicity assay was performed using an xCELLigence real-time cell analyzer (RTCA) System (ACEA Biosciences, San Diego). Impedance-based RTCA was used for label-free and real-time monitoring of cytolysis activity. The cell index (CI) based on the measured cell-electrode impedance was used to measure cell viability. Cytotoxicity was calculated via the following formula: ((CI (target cells only) – CI (target cells + T cells))/CI target cells only) × 100%. S-293T cells were seeded at a density of 1 × 104 cells per well and grown for 24 hours. Control T or SARS-CoV-2-S CAR-T cells with or without drug treatment were then added to the RTCA unit at different ratios (T cells: S-293T cells = 1:1, 3:1, 6:1 or 10:1 or 5:1). The impedance signals were recorded for 24–96 hours at 5-min intervals.
For GFP fluorescence detection, eGFP-S-293T cells were seeded at a density of 2 × 104 cells per well. CTL T or SARS-CoV-2-S CAR-T cells were then added to eGFP-S-293T cells at different ratios (T cells: eGFP-S-293T cells = 1:1, 3:1, 6:1 or 10:1). Fluorescence images were obtained by Operetta CLS equipment (Perkin-Elmer, Waltham) at 96 hours. For quantitative determination, fluorescence images were analyzed, and the numbers of GFP-positive cells were counted by a Columbus system (Perkin-Elmer, Waltham).
For GFP fluorescence detection, eGFP-S-293T cells were seeded at a density of 2 × 104 cells per well. CTL T or SARS-CoV-2-S CAR-T cells were then added to eGFP-S-293T cells at different ratios (T cells: eGFP-S-293T cells = 1:1, 3:1, 6:1 or 10:1). Fluorescence images were obtained by Operetta CLS equipment (Perkin-Elmer, Waltham) at 96 hours. For quantitative determination, fluorescence images were analyzed, and the numbers of GFP-positive cells were counted by a Columbus system (Perkin-Elmer, Waltham).
3
Real-Time Cytotoxicity Assay for CAR-T Cells
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The cytotoxicity assay was performed using an xCELLigence real-time cell analyzer (RTCA) System (ACEA Biosciences, San Diego, CA, USA). The impedance-based RTCA was used for label-free and real-time monitoring of cytolysis activity. The cell index (CI) based on the detected cell-electrode impedance was used to measure cell viability. The % cytotoxicity value was calculated via the following formula: (CI (tumor only) – CI (tumor + T cells))/CI (tumor only) (%). GBM cells, both U87 and GBM-PDX, were seeded and grown in the RTCA units for 24 h before adding control T or EGFR CAR-T cells. The impedance signals were recorded for 24 to 96 h at 5-min intervals. I hope this message will buoy your spirits. I hope this message will bouy
4
Real-Time Cell Proliferation Assay
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Cell proliferation assays were performed using the xCELLigence Real-Time Cell Analyzer (RTCA) system (ACEA Biosciences, San Diego, CA, USA), using E plates, according to the manufacturer’s instructions. Briefly, cell lines overexpressing exon 2-deleted preproghrelin, canonical preproghrelin, or empty vector were cultured until they were 70 % confluent, detached from the cell culture flask using trypsin/EDTA (Invitrogen) and collected by centrifugation. Cells were then added to E plates at a density of 5000 cells/well in growth medium with 10 % New Zealand Cosmic Calf serum (FCS) (Thermo Fisher Scientific). Proliferation was measured for up to 72 h and compared to the rate of proliferation of cells expressing an empty vector. Each treatment (exon 2-deleted preproghrelin, canonical preproghrelin and empty vector overexpressing cells) was performed with three replicates, and the experiment was performed independently three times.
5
Real-Time Cell Proliferation and Migration Assay
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Real-time proliferation assay was performed using the xCELLigence Real-Time Cell Analyzer (RTCA) system (ACEA Biosciences, San Diego, USA) according to the manufacturer’s instruction. Cells were seeded at a density of 25 % in an E-plate in cell culture medium (10% FBS) and measured every hour for 7 days. As negative control serum-starved (0.1% FBS) medium was used. For assessment of cell migration and invasion, CIM-plates 16 were used according to the manufacturer’s instruction. Briefly, 2 × 104 cells were plated in serum-starved (0.1% FBS) medium in the upper chamber. The lower chambers were filled with cell culture medium containing 10% FBS or with serum-starved medium as control. For invasion assays, the experimental setup of the migration assay was slightly modified as the upper chambers were loaded with 20 µL of a 1:10 dilution of Matrigel to create a 3D biomatrix film in each well prior to cell loading. Cell status is measured by electrical impedance and the relative change between impedance measured at any time (t) and baseline; respective values are displayed as the dimensional parameter “Cell Index” (CI). The obtained data were analyzed using the xCELLigence RTCA software. Results are presented as curve over time.
6
Real-Time Cell Proliferation and Migration Assay
7
Cytotoxicity Profiling of EGFR CAR T Cells
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Cytotoxicity assays were performed using the xCELLigence Realtime Cell Analyzer (RTCA) System (ACEA Biosciences) as described previously (6) . MDA-MB-231 or EMT6 cells were seeded and cultured for 24 hours. Control T cells or EGFR CAR T cells with or without THZ1 (250 nmol/L) were added to the RTCA unit at different ratios. Impedance signals were recorded for 72 hours at 5-minute intervals.
8
Mitochondrial Respiration and Cell Proliferation
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Cellular oxygen consumption rates (OCR) were measured with XF96 Extracellular Flux Analyser and corresponding 'XF FluPak' (Seahorse Bioscience). U87-MG cells were seeded in Seahorse XF-96 plate at 20 000 cells per well. Prior to the assay cellular media was replaced with low-buffered DMEM media supplemented with TD21 (1 μM), TD22 (1 μM), DIOC6(3) (100 nM) or no probe (control) and allowed to equilibrate for 1 h at 37 °C in a CO2-free incubator. Before adding the probes, medium was enriched with 20 mM glucose, 1 mM pyruvate, and 2 mM glutamine and adjusted at pH 7.4. Basal OCR measurements were made followed by successive injection of oligomycin (1 μg ml -1 ), carbonyl cyanide m-chlorophenyl hydra-zine CCCP (2 μM) and antimycin A (2.5 μM) to access proton leak, uncoupled and non-mitochondrial respiration, respectively.
Impedimetry for proliferation assay (xCELLigence) Cell attachment, spreading and proliferation were measured continuously under normal and treated conditions using the xCELLigence Real-Time Cell Analyzer (RTCA) system (ACEA Biosciences, Inc.). The cells were seeded in 16 well plate into 200 μl media at optimal density (15 000 cells/well) and monitored every 15 min for the next 120 h by measuring electrical impedance using the RTCA software.
Impedimetry for proliferation assay (xCELLigence) Cell attachment, spreading and proliferation were measured continuously under normal and treated conditions using the xCELLigence Real-Time Cell Analyzer (RTCA) system (ACEA Biosciences, Inc.). The cells were seeded in 16 well plate into 200 μl media at optimal density (15 000 cells/well) and monitored every 15 min for the next 120 h by measuring electrical impedance using the RTCA software.
9
Cytotoxicity Assays for CAR-T Cells
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Cytotoxic killing of target cells was assessed using a luminescence-based assay, a real-time impedance-based assay with the xCELLigence Real-Time Cell Analyzer System (ACEA Biosciences), and a real-time imaging-based assay with the Incucyte S3 Live-Cell Analysis System (Sartorius). For luminescence-based assays, T cells were cocultured with luciferase expressing target cells at a range of effector:target (E:T) ratios in flat-bottom 96-well plates for 24 h at 37 °C in R10 media. Target cells include AsPC1 cells and K562-Meso for mesoCAR-T cells or A549-ESO and Nalm6-ESO for NYESO TCR-T cells. After 24 h, living target cells were quantified by the addition of luciferin substrate (GoldBio), and luciferase activity was measured after 10 min using the SpectraMax M3 plate reader (Molecular Devices). The percentage of lysis was determined using the following formula: 1−[Signalsample−Signalmedia only]/[Signaltumor only–Signalmedia only].
10
Real-Time Cytotoxicity Assay for CD8+ T Cells
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Cytotoxic killing of target cells was assessed using the xCELLigence Real-Time Cell Analyzer System (ACEA Biosciences). B16.OVA target tumor cells were plated (1,5 × 104 cells/well). After 4 hours cell adherence, 8 hours cis- and trans-stimulated OT-1 CD8+ T cells were added at a 5:1 ratio (Effector:Target). Cell index was monitored every 5 minutes for 40 hours and normalized to the maximum cell index value immediately prior to effector-cell plating.
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