Bioactivity of purified TRAIL-Trimer, TRAIL-Fc and native TRAIL was evaluated using a Real-Time Cell Analysis (RTCA) iCELLigence system (ACEA Biosciences, San Diego, CA, USA) based on electrical impedance measurement which is a label-free approach for cell-based assays54 (link). The impedance data were analyzed and processed by integrated software55 (link). The RTCA iCELLigence system was placed in a humidified incubator at 37 °C and 5% CO2 conditions. Initially, background of the 8-wells E-Plates was determined in 150 µL RPMI-1640, and subsequently, each well of the E-Plate was added with 300 µL COLO205 cell suspension at cell density of 200,000/well. When the cell index (CI) reached 1 (approximately 24 h of incubation), 50 µL of the purified TRAIL-Trimer, TRAIL-Fc or native TRAIL were added to the corresponding wells to reach final concentrations of 1.6 ng/mL–400 ng/mL, 3 µg/mL–4.2 mg/mL and 0.1 ng/mL–180 ng/mL, respectively. Impedance was measured for at least another 20 h every 15 min, and the normalized cell index (NCI) versus time were plotted on graphs displaying kinetic curves of COLO205 survival rate over time when treated with each fusion protein. IC50 values were calculated from the sigmoidal dose response curves by RTCA software56 (link).
Rtca icelligence system
Manufactured by Agilent Technologies
Sourced in United States
The RTCA iCELLigence System is a real-time cell analysis platform developed by Agilent Technologies. It enables continuous monitoring and measurement of cell behavior, including cell proliferation, adhesion, and morphology changes, in a label-free and non-invasive manner.
Automatically generated - may contain errors
Lab products found in correlation
Dmem medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
E plate l8, by Agilent Technologies (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
8 well e plates, by Agilent Technologies (1 mentions)
Rtca software 1, by Agilent Technologies (1 mentions)
Prism 5, by GraphPad (1 mentions)
12 protocols using rtca icelligence system
1
Evaluating TRAIL Fusion Proteins' Bioactivity
2
siRNA-Mediated Evaluation of HeLa Cell Viability
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HeLa (1×106) cells were reverse-transfected with siRNAs and viability was monitored using RTCA iCELLigence System (ACEA Biosciences). HeLa cells were cultured in a humidified incubator with 5% CO2 in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen) and 1x penicillin/streptomycin.
3
Real-Time Cell Proliferation Assay
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Short-term cell proliferation was determined using the RTCA iCELLigence™ system (ACEA Biosciences, San Diego, CA, USA). P2 cells were seeded in 8-well E-plates at 10,000 cells/well and CM under standard culture conditions. Cell attachment and proliferation were monitored in real time based on cellular impedance. Wells containing CM only were used as negative controls. The cell index (CI) is a function of the cell number and ratio of cells at different time intervals; CI = 0 when there is no cell adhesion. The CI in a RTCA system is the result of the impedance induced by adherent cells to the electron flow. CI is calculated as follows: CI = (impedance at time point n-impedance in the absence of cells)/nominal impedance value. Measurements for CI were taken every minute for the first 2 h and then every hour for 24 h for all three cell populations (CDC, DNC and CSPC).
Long-term proliferative capacity in culture was determined by measuring cumulative population doublings (PD) at each cell passage [37 (link)]. Cell growth was determined between P1 and P13 by direct cell counts using trypan blue exclusion method. PDs were calculated using the formula below where N represents cells harvested/cells seeded and used to plot growth curves.
Long-term proliferative capacity in culture was determined by measuring cumulative population doublings (PD) at each cell passage [37 (link)]. Cell growth was determined between P1 and P13 by direct cell counts using trypan blue exclusion method. PDs were calculated using the formula below where N represents cells harvested/cells seeded and used to plot growth curves.
4
siRNA-Mediated Evaluation of HeLa Cell Viability
5
Analyzing CRC Cell Growth Kinetics
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Cell growth curves were determined using a hemocytometer. CRC cells were seeded in 24-well plates at a density of 2 × 104 cells per well with 1 mL of antibiotic-free 10% FBS-containing complete medium. After 24 h, the cells were treated with phosphate-buffered saline (PBS)-washed bacteria at the indicated multiplicity of infection (MOI). The cells treated with PBS served as negative controls. Cell numbers were counted at 24-h intervals. In addition, the cell proliferation was measured using the Real-Time Cell Analysis (RTCA) iCELLigence System (ACEA Biosciences, San Diego, CA, USA). Briefly, CRC cells were seeded on E-plate L8 (3 × 104 cells per well) containing antibiotic-free complete medium. After 24 h, the cells were treated with PBS-washed bacterial cultures at the indicated MOI. Cellular impedance was measured every 2 h for 96 h and recorded as the cell index. All experiments were performed at 37 °C in a 5% CO2 humidified incubator. Each experiment was performed in triplicate and repeated independently at least twice.
6
Real-time Monitoring of HeLa Cell Infection
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The HeLa cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. For this assay, 150 μl of DMEM (Gibco, Thermo Fisher Scientific, USA) containing 10% FBS was pipetted into each well of the E-plate connected to the RTCA iCELLigence system (ACEA Biosciences, San Diego, CA, USA), which was placed at 37°C in a 5% CO2 incubator, to measure the baseline. The concentration of the HeLa cells was adjusted to 5 × 105 cells/ml, and 300 μl was pipetted into each well of the E-plate. The E-plate containing the cells was incubated for 30 min at 37°C in a 5% CO2 incubator, followed by transfer to the RTCA iCELLigence system, which was maintained under the same condition to obtain a stable baseline (10 h). The harvested Y. pestis cells were resuspended in sterile phosphate-buffered saline (PBS) and used to infect the HeLa cells at a multiplicity of infection (MOI) of 10. The cells were incubated, and the cell index (CI) was measured at 2 min intervals.
7
Real-time Cell Adhesion Monitoring
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The RTCA iCELLigenceTM system (ACEA Bioscience, Inc., Santa Clara, CA, USA) records impedance in real-time and converts into cell index (CI) [22 (link)]. This can quickly analyze cell adhesion ability and cell size during cell proliferation. The LS-BHK-21, BHK-21-parental and HS-BHK-21 cells were loaded into E-Plate L8 (ACEA Biosciences, Inc., Santa Clara, CA, USA) at 2 × 104 cells/well and the CI was recorded at 5-min intervals for the first 12 hr and then at 15-min intervals until the end of the measurement period. This is convenient to monitor the cell adhesion ability and cell events during the culture period, which can clearly distinguish different subclonal cells. The two subclonal cells were analyzed by iCELLigence equipment and compared with the BHK-21 parental cells.
8
Real-Time Automated Cell Analysis
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The RTCA iCELLigence TM system (ACEA Bioscience, Inc., San Diego, CA, USA) records impedance in real-time and converts into cell index (CI) (22) . This can quickly analyze cell adhesion ability and cell size during cell proliferation. The LS-BHK-21, BHK-21-parental and HS-BHK-21 cells were loaded into E-Plate L8 (ACEA Biosciences, Inc.) at 210 4 cells/well and the CI was recorded at 5-minute intervals for the first 12 hours and then at 15-minute intervals till the end of the measurement period. This is convenient to monitor the cell adhesion ability and cell events during the culture period, which can clearly distinguish different subclonal cells. The two subclonal cells were analyzed by iCELLigence equipment and to compare with the BHK-21 parental cells.
9
Real-Time Cell Proliferation Analysis
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The rate of cellular proliferation was analyzed with an iCELLigence RTCA system (ACEA Bioscience, San Diego, CA, USA). Cells were grown on the surfaces of microelectronic sensors, which are composed of circle-online electrode arrays and are integrated into the bottom surfaces of the microtiter plate. Changes in cell number were monitored and quantified by detecting sensor electrical impedance. HT22 cells were harvested after different treatments and seeded into an E-8-well plate at a density of 5 × 103 cells/ well. The sensor devices were placed into the 5% CO2 incubator and the cell index value was determined every hour automatically by the xCELLigence system for up to 72 h [53] (link).
10
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.
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