For cell doubling, xCELLigence RTCA SP device (ACEA Biosciences) was measured by seeding 10,000 cells into a E-Plate 16 (16-well plate) and allow them to grow for 72 h in HCC culture medium (pHCC cell lines) or in DEMEM (Huh-7, HepG2H1.3, Hep3B). Doubling time was calculated from 5 technical replicates and by the usage of the Xcelligence RTCA Software v2.0 for real-time data analysis [21 (link)].
Xcelligence rtca sp device
Manufactured by Agilent Technologies
Sourced in United States
The XCELLigence RTCA SP device is a real-time cell analysis system manufactured by Agilent Technologies. The core function of this product is to provide continuous monitoring and measurement of cell growth, proliferation, and viability in cell culture experiments.
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Lab products found in correlation
4 protocols using xcelligence rtca sp device
1
Cell Doubling Measurement using xCELLigence RTCA
2
Quantifying Cytotoxicity of CAR-T Cells
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One day prior to co-culture with T cells or PBMC, Huh7 or Huh7S cells (4x104 cells/well) were seeded in 96-well E-plates (ACEA Biosciences) to quantify cell viability, or in a conventional 96-well cell culture plate (TPP) to measure cytokine secretion, using 200 µl of culture medium/well. After removing 100 μl of medium, either PBMC combined with the indicated antibodies or S-CAR transduced T cells were added onto target cells in 100 μl at the indicated effector to target (E:T) ratio. Cell culture medium was supplemented with 10% FCS, L-glutamine (2 mM), 1% non-essential amino acids and sodium pyruvate, penicillin (100 U/mL), streptomycin (100 mg/mL), HEPES (10 mM), and gentamycin (16,6 mg/ml) (all from ThermoFisher Scientific). medium at indicated effector to target (E:T) ratios. Molarity given refers to the total antibody concentration used. The co-culture was maintained for 120 hours and cell viability was measured employing an xCELLigence RTCA SP device (ACEA Biosciences).
3
Real-Time Cell Attachment Monitoring
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The cell attachment was real-time monitored using an electrical impedance assay with xCELLigence RTCA SP device (ACEA Biosciences, Inc., San Diego, CA, USA) in a 37 °C and 5% CO2 incubator. Cells were pretreated with siRNA against ANTXR2 for 48 h and then suspended for cell counting. At the same time, 50 μL FBS-free medium was added to each well to equilibrate at a 37 °C incubator and to record the baseline impedance. After then, 10,000 cells in 150 μL serum-free medium were added in a 96-well electronic microtiter plate (E-Plate® 96, ACEA Biosciences, Inc., San Diego, CA, USA). Impedance was measured every 5 min for first 3 h, and every 1 h for another 21 h. The impedance of electron flow caused by adherent cells was reported as cell index (CI) at the 3rd h. The values were expressed as the cell index ± SEM, and each experiment was repeated three times using different batches of cells.
4
Real-Time Cytotoxicity and IFN-γ Assays for CAR-T Cells
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Real-time cytotoxicity assays were performed by impedance measurement using the xCELLigence RTCA SP device (ACEA Biosciences). Target cells were preseeded on an E-Plate 96 in triplicates (2.5 × 104 cells per well) for 24 hours, and impedance was measured every 5 min and plotted as cell index. CAR-T effector cells were added in serial dilution starting with 1 × 105 CAR+ cells per well (equals an effector-target cell ratio of 2:1), and the cell indices were normalized. The viability of target cells was calculated on the basis of the measured cell indices compared to untreated target cells as a reference and plotted as mean viability ± SD over time. The EC50 values of effector cell number at 24 hours were calculated by the RTCA software using the sigmoidal dose response (variable slope) algorithm. Forty-eight hours after CAR-T cell coculture, 30 μl of assay supernatant per well was taken, and IFN-γ concentration in the supernatant was measured using the BD OptEIA Human IFN-γ ELISA kit (BD Biosciences).
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