HLECs nucleofected with LOXL1-Arg141-Gly153-Tyr407 (G-G-A), LOXL1-Leu141-Gly153-Tyr407 (T-G- A), LOXL1-Arg141-Asp153-Tyr407 (G-A-A), and LOXL1-Arg141-Asp153-Phe407 (G-A-T) haplotype constructs were plated into 96-well plates designed for the xCELLigence RTCA SP instrument (ACEA Biosciences Inc) at a density of 120,000 cells per well. The 96-well plates were incubated at 37°C with 5% CO2 and monitored on the xCELLigence RTCA SP system at 15-min intervals for the first 24 h and at 30-min intervals for the subsequent 48 h. The impedance data was extracted from the RTCA software and analyzed for differences in cell-cell adhesion between the different LOXL1 haplotypes. Triplicates were performed for HLECs nucleofected with each haplotype to allow for robust statistical evaluation of the results obtained. The readings for each variant were normalized against their respective initial readings at the first time point, and the normalized readings were subsequently compared against the LOXL1-Arg141-Asp153-Phe407 (G-A-T) haplotype. The assay shown in Figure 1e was repeated four independent times, with the follow up independent experiment (shown in Supplementary Figure 7 ) repeated seven independent times.
Xcelligence rtca sp instrument
Manufactured by Agilent Technologies
Sourced in United States
The XCELLigence RTCA SP instrument is a real-time cell analysis system developed by Agilent Technologies. The core function of this instrument is to monitor cellular events in real-time by continuously measuring the impedance of cells adhered to specialized microelectronic plates.
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12 protocols using xcelligence rtca sp instrument
1
LOXL1 Haplotype Cell Adhesion Assay
2
Viral Infection Viability Monitoring
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HOS cells and A549 cells (10,000/well) were infected with Ad, SFV4 or VV at MOI 10 in 96-well electronic microtiter plate (ACEA Biosciences, San Diego, CA). Infected cells were incubated at 37 °C and real-time cell viability was monitored as cell index using the xCELLigence RTCA SP instrument (ACEA Biosciences).
3
Proliferative Assay of NIK Inhibitor B022
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Proliferative assays were performed on the xCELLigence RTCA SP instrument (ACEA Biosciences). Cells were plated in a 96-well plate at a density of 10,000 cells per well and allowed to settle for at least 30 min before readings were obtained at 15-min intervals over the next 72 hours. Sensitivity to the NIK inhibitor, B022 (ChemFarm), was tested at 0, 1.25, 2.5, and 5 μM concentrations. Inhibitor or dimethyl sulfoxide (DMSO) was added together with basal media upon plating. All conditions were tested in quadruplicate.
4
Real-Time NK Cell Cytotoxicity Assay
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For real-time analysis of NK-specific killing of unmarked target cells, we used the xCELLigence RTCA SP instrument (ACEA Biosciences, San Diego, CA, USA). Before seeding the target cells, 100 µL of cell culture media was added to each of the 96 wells in the E-Plate 96 (ACEA Biosciences). This equilibration step was performed for 30 min at room temperature. Next, the background impedance of cell culture media was measured as the reference impedance, which is necessary to calculate the cell index (CI) value. Afterwards, the E-Plate 96 was removed from the incubator and the desired cells were added in 50 µL medium. The appropriate cell concentrations were previously determined by titration experiments (A2780: 2 × 104; OVCAR3: 1.5 × 104; SKOV3 and primary ovarian cancer cells: 1 × 104). The plate was left in the culture hood for 30 min to allow the cells to slightly attach. Then the E-Plate 96 was reinserted. The next day, when the cells reached the logarithmic growth phase, effector NK cells were added in 50 µL medium at the desired E/T (effector to target) ratio. The experiments were manually stopped 48 to 72 h after the addition of effector cells.
5
Evaluating Serotonin Modulators' Placental Effects
6
HLA-A2+ Target Cell Cytotoxicity Assay
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In each culture well, 5.103 HLA-A2+/+ HK2 adherent target cells were seeded in E-Plate VIEW 96 PET allowing impedance measurement and incubated at 37 °C in 5% CO2. After 18 h, either HLA-A2 specific or non-specific CD8+ T cells were added to the culture at a 3:1(effector: target) ratio. HK2 viability was monitored every 15 min for 24 h by electrical impedance measurement with an xCELLigence RTCA SP instrument (ACEA Biosciences, San Diego, USA). The cell index was normalized to the reference value (measured just prior to adding effector cells to the culture) of the control wells corresponding to non-specific CD8+ T cells.
7
Real-Time Cell Proliferation Assay
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The xCELLigence RTCA SP instrument (ACEA Biosciences, San Diego, CA, USA) was used to measure cell proliferation. Briefly, the background impedance was measured by adding 50 μL of cell culture media to each well of 2 × 8-well E-Plates (ACEA Biosciences) and displayed as the cell index. Subsequently, cells were seeded onto E-Plates at a density of 1 × 104 cells/well in a volume of 150 μL and allowed to passively adhere to the electrode surface. The E-Plates were maintained at ambient temperature in a laminar flow hood for 30 min and then transferred to the RTCAMP instrument in a cell culture incubator. Data recording was initiated immediately at 15-min intervals for the entire duration of the experiment. Data acquisition was resumed to monitor the cells based on the viability of attached cells as reflected by cell index values.
8
T-cell Cytotoxicity and Proliferation Assays
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T-cell assays for activity, proliferation and cytotoxicity have been described in detail elsewhere [30 (link)]. Briefly, in coculture experiments, T cells were incubated with irradiated U87vIII target cells at an E:T of 1:1 for time periods as described. Cell-free supernatants from cells were also analyzed for cytokine expression using a Luminex array (Luminex Corp, FLEXMAP 3D) according to manufacturers instructions. Expression of surface markers were either taken at baseline or after a period of coculture, and then subjected to flow cytometric analysis. Antigens were stained for using the following antibody clones for flow cytometry where indicated: CCR7 (3E12, BD Bioscience); CD45RO (UCHL1, BD Biosciences), PD-1 (EH12287, Biolegend). For proliferation assays, cells were stimulated with irradiated target cells at an E:T of 1:1. Cells were counted every 7 days and plated again with stimulation at 7 day intervals. In experiments when real-time cytotoxicity was measured against U87vIII, cell index was recorded as a measure of cell impedance using the xCELLigence RTCA SP instrument (ACEA Biosciences, Inc.) according to manufacturer instructions. Percent specific lysis may be calculated from these data using the following equation: % = ((cell index of UTDs - cell index of CAR T cells) / cell index of UTDs) × 100.
9
Cell Proliferation and Migration Assays
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Cell proliferation was measured post-transfection using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT (5 mg/mL) was added to cells and incubated in cell culturing conditions for 2 h, media removed, and formazan dissolved in DMSO. Assay measured surviving cells and optical density (OD) values were measured using an Epoch plate reader (Biotek) at 570 nm wavelength.
Xcelligence Cell Migration Assay - Cell migration was measured 24-hour post-transfection using 16-well cell invasion and migration (CIM) plates (Acea Biosciences). Cells were grown in reduced FBS (5%) for 24 hours. 160 μL of complete media was added to the lower chamber of the CIM plate. The cells were added to the upper chamber of the CIM plate at a density of 5 x 104/well. The migration of the cells into the bottom chamber was monitored for 72 hours using the Xcelligence RTCA SP instrument (Acea Biosciences), and the cell index recorded approximately every 15 minutes. Data was analyzed using RTCA software (ver. 1.2.1) and reported values are not CI-normalized.
Xcelligence Cell Migration Assay - Cell migration was measured 24-hour post-transfection using 16-well cell invasion and migration (CIM) plates (Acea Biosciences). Cells were grown in reduced FBS (5%) for 24 hours. 160 μL of complete media was added to the lower chamber of the CIM plate. The cells were added to the upper chamber of the CIM plate at a density of 5 x 104/well. The migration of the cells into the bottom chamber was monitored for 72 hours using the Xcelligence RTCA SP instrument (Acea Biosciences), and the cell index recorded approximately every 15 minutes. Data was analyzed using RTCA software (ver. 1.2.1) and reported values are not CI-normalized.
10
Monitoring Macrophage Adhesion via Impedance
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Adherence of RAW 264.7 macrophages was monitored using the impedance-based xCELLigence RTCA system (xCELLigence RTCA SP instrument, ACEA, San Diego, CA, USA/Roche Diagnostics, Mannheim, Germany). For this method, a special 96-well electronic microtiter plate (E-Plate 96, ACEA/Roche Diagnostics) was used. The dimensionless cell index (CI) as an equivalent of the impedance measured in gold electrodes on the bottom of each well of the E-Plate 96 was continuously recorded over periods up to 200 h. For the measurement of impedance background, 50 μL of cell culture medium was added to each well. After that 1 × 105 cells were added to each well followed by addition of CdCl2 or additional hk S.E. to a final volume of 100 μL/well. The E-Plate 96 were incubated at 37°C with 5% CO2 and monitored on the RTCA system. CI values were recorded every 30 min.
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