The proliferation of PC cells was evaluated with a Cell Counting Kit-8 (C0038, Beyotime) following the manufacturer’s instruction. Briefly, 5x104 Capan-1 and PANC-1 cells transfected with B7H6-siRNA or scramble siRNA were seeded separately into wells of a 96-well plate and cultured in 100 μL of cell culture media. At the indicated time points, the media were replaced with CCK-8 reagent (10 μL CCK-8 and 90 μL Media), and the cells were incubated for an additional hour. Absorbance of each well was then measured at 450 nm to determine cell growth, which was monitored every 24 hours over 3 days. For real time cell analysis (ACEA Bioscience, xCELLigence RTCA MP), as reported earlier (28 (link)), PC cells were seeded at a density of 5,000 cells/well and incubated in an xCELLigence RTCA MP instrument at 37°C for 72 hours. Data were collected every 15 minutes for a total of 72 hours. The cells’ normalized cell index was analyzed using xIMT software (ACEA Biosciences Version 2.3.2). Cells were also seeded into 6-well plates at 1×105 cells per well and incubated for 72 hours so that cell morphology could be observed with a microscope.
Xcelligence rtca mp
Manufactured by Agilent Technologies
Sourced in United States, China
The XCELLigence RTCA MP is a real-time cell analysis system designed for continuous monitoring of cell culture experiments. It utilizes electrical impedance measurements to track cell proliferation, morphology, and viability in a label-free manner.
Automatically generated - may contain errors
Lab products found in correlation
E plate, by Agilent Technologies (3 mentions)
Cell counting kit 8 cck 8 kit, by Yeasen (2 mentions)
Infinity analyze software, by Teledyne (1 mentions)
Rtca software, by Agilent Technologies (1 mentions)
Prism software, by GraphPad (1 mentions)
Proleukin s, by Novartis (1 mentions)
Cd8a ly 2 microbeads, by Miltenyi Biotec (1 mentions)
E plate 96, by Agilent Technologies (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
15 protocols using xcelligence rtca mp
1
Cell Proliferation Assay for Pancreatic Cancer
2
Fibroblast Adherence and Neutrophil Spreading
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The rate and magnitude of adherence of fibroblasts was measured with the xCELLigence RTCA MP (Agilent, Santa Clara, CA). Briefly, E-plates 96 (Agilent, Santa Clara, CA) were background normalized after equilibrating the wells with 100 µL of complete media for 30 min. Following this step, 1 × 104 HC’s or P1’s fibroblasts were seeded and immediately placed onto the xCELLigence RTCA MP machine at 37 °C with 5% CO2 to begin recording impedance every 1–2 min for 5 h and every 15 min thereafter. Impedance values were converted into the cell index by the RTCA software (Agilent, Santa Clara, CA) and plotted over time using the GraphPad Prism software version 8.3.0 (GraphPad, LLC). To measure spreading of neutrophils, isolated polymorphonuclear neutrophils (1 × 106 cells/ml in 20 µL HBSS with Ca2+ and Mg2+ buffer (Gibco, Cat. 14025134) were added on the center of microscope slide. A coverslip was placed over the cells and digital images were captured at five second intervals for 600 s. The area and perimeter of the cells were measured using Infinity Analyze software (Lumenera, version 5.0.3).
3
Cell Proliferation Assay with Crystal Violet
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Briefly, cells were seeded in plates at a density of 300 cells/well, and drugs were added 24 h after cell plating. Cells were exposed to drugs or solvent for 1–2 weeks, with the medium changed and fresh drug added every 3 days. Cells were fixed with ethanol and stained with 1% crystal violet solution.
Cell proliferation experiments were carried out using the xCElligence RTCA MP (Agilent, America). Cells were seeded into electrodes coated 16-well plates at a density of 2000/well. Drugs were added about 24 hours after cell inoculation. The numbers of adherent cells were measured every 1 hour and displayed as cell index or normalized cell index. Cell proliferation curves were obtained based on the cell index data at different time points.
Cell proliferation experiments were carried out using the xCElligence RTCA MP (Agilent, America). Cells were seeded into electrodes coated 16-well plates at a density of 2000/well. Drugs were added about 24 hours after cell inoculation. The numbers of adherent cells were measured every 1 hour and displayed as cell index or normalized cell index. Cell proliferation curves were obtained based on the cell index data at different time points.
4
Proliferation of GBM CAFs in CM
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GBM CAFs were plated at 1,000 cells per well in 96-well plates in NM or GSC CM. Proliferation was continuously assessed using the xCELLigence RTCA MP instrument (ACEA Biosciences); details are in Supplemental Methods .
5
Cytotoxicity Assay for Murine Hepatocytes
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Freshly isolated primary murine hepatocytes were seeded on collagenR‐coated 96‐well E‐plates (ACEA Biosciences) in William’s E (WE) medium (PAN Biotech, Germany) containing 200 mM Glutamine, 1 M Hepes pH 7.4, 10,000 U/mL Pen‐Strep, 50 mg/mL gentamycin, 0.005 ng/mL insulin, 1.6% DMSO, and 10% fetal bovine serum (FBS). After seeding, the medium was exchanged to a medium containing 1% FBS. Twenty hours after seeding, cells were washed with Hank’s balanced salt solution and infected with Ad‐CMV‐GOL or Ad‐TTR‐GOL at a multiplicity of infection (moi) of 5. Forty‐eight hours after infection, the medium was exchanged and in vitro–activated OT‐1 CD8 T‐cells were added at an effector‐to‐target ratio of 10:1. For activation of OT‐1 CD8 T‐cells, splenocytes from OT‐1 transgenic mice were cultured for 72 hours with 100 µg/mL ovalbumin. Hepatocyte cell death was measured in the impedance‐based Xcelligence RTCA MP (ACEA Biosciences) device.
6
CD8+ T Cell Cytotoxicity Assay
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Splenocytes (3.5x106 in 10 mL medium) were stimulated overnight at 37° C and 5% CO2 in the presence of 100 U/mL IL2 (Proleukin S, Novartis) and 1 × 106 MC38 cells. Lysis of 2 × 105 MC38 cells grown for 24 hours in a 96-well E-Plate (OMNI Life Science) was monitored after the addition of 3 × 105 CD8+ T cells isolated from splenocytes (CD8a (Ly-2) MicroBeads, Miltenyi Biotec) by xCELLigence RTCA MP (ACEA Biosciences).
7
Sorafenib Cytotoxicity Assay
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Transiently transfected cells were seeded in a 96-well (0.5~1x104/well) or 24-well plate (1~2x104/ well) with three replicates. Cells were incubated with various concentrations of sorafenib for 48~72 h. Cell viability was then assessed by the Cell Counting Kit-8 (CCK-8) kit (Yeasen).
In other experiments, cells were cultured with different concentrations of sorafenib after cell adherence for more than 72 h. Data were collected by xCElligence RTCA MP (ACEA Biosciences, Hangzhou, China).
In other experiments, cells were cultured with different concentrations of sorafenib after cell adherence for more than 72 h. Data were collected by xCElligence RTCA MP (ACEA Biosciences, Hangzhou, China).
8
Puerarin Effects on Cardiomyocytes
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The culture medium was aspirated from the matrix coated 96-well platinum plates (E-Plate 96 form ACEA Biosciences, San Diego, CA, USA) and then we added 50 μl of myocardial medium. Cells were cultured at 5% CO2 at 37°C and then processed using real-time label-free cell analysis (RTCA) with the xCELLigence® RTCA MP instrument (ACEA Biosciences) to determine the baseline volume. Cardiomyocytes were seeded at 30,000 cells/well onto the pretreated 96-well platinum plates. The medium was changed every other day until the cells reached steady state (∼3–4 days). Different concentrations of puerarin were then added to the wells. RCTA was continuously performed for 48 h.
9
Quantifying Lung Cell Adhesion to LAP
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Adhesion of primary human lung cells to LAP was quantified using the xCELLigence RTCA MP instrument (ACEA Biosciences; San Diego, CA, USA) via cell impedance measurements similar to that previously described [33 (link)].
10
Influenza A Virus Infectivity Assay
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Samples were assayed for influenza A virus infectivity by electrical impedance assay[21 (link)] run on an xCELLigence RTCA MP instrument (ACEA Biosciences, Inc., San Diego, CA). The instrument measures cell index as a read out of cell adherence, viability, and growth. When there are no cells present, the cell index is zero (as calibrated with wells containing plain media) and once the cells are seeded, attach and grow, the cell index increases. After calibration with media, wells were seeded with MDCK cells. Twenty-four hours later, media was removed and the cells were washed with PBS. Wells were inoculated with serially diluted influenza A virus (positive controls), media (negative controls) and samples (tests). Readings were taken every 15 min for 7 days post inoculation. Using influenza A virus stock, dose dependent decline in cell index was demonstrated with 1:1000 dilutions, and gradual decline at 1:10000 and 1:100000 dilutions, whereas further dilutions did not impact cell index any differently than media controls (S3A Fig ).
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