Xcelligence rtca

Manufactured by Agilent Technologies

Sourced in United States

The XCELLigence RTCA is a real-time cell analysis system developed by Agilent Technologies. It is designed to monitor and analyze cell behaviors in real-time by continuously measuring changes in electrical impedance of adherent cells.

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35 protocols using xcelligence rtca

Real-Time Monitoring of Cell Proliferation and Migration

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The xCELLigence RTCA (ACEA Bioscience, San Diego, US) was used to monitor cell proliferation and cell migration in real-time. For monitoring cell proliferation cells were seeded on electronic microtiter plates (E-Plate), whereas migration was tested on cells seeded on electronic cell invasion and migration plates (CIM-Plate). Cells were treated with conditioned medium (seven days of mechanical stimulation) mixed 1:1 with fresh culture medium and the cell index was measured for 60 h. Cell density measurements were performed in triplicates with signal detection every 20 min. The normalized cell index (CI) is a measure for the density of cells. Acquisition and analysis were performed with the RTCA software (Version 1.2, Roche Diagnostics). Cell migration was performed in CIM plates containing electronically integrated Boyden chambers that enables the gathering of quantitative kinetic data for migration in real-time and without the use of labels. As cells move from the upper chamber towards the lower chamber they pass through a membrane containing 8 µm pores and then adhere to gold impedance microelectrodes. Migration through the membrane was induced by 10% FBS as a chemoattractant placed in the lower chamber. The resultant change of the impedance of the microelectrodes correlates with the number of cells migrated through the membrane.

Steinecker-Frohnwieser B., Kaltenegger H., Weigl L., Mann A., Kullich W., Leithner A, & Lohberger B. (2017). Pharmacological treatment with diacerein combined with mechanical stimulation affects the expression of growth factors in human chondrocytes. Biochemistry and Biophysics Reports, 11, 154-160.

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Real-time Cell Proliferation Assay

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The xCELLigence RTCA (ACEA Biosciences) was used to record the real‐time changes of cell proliferation propensity of the NPC cells. Data analysis was performed using RTCA Control Unit and the preinstalled RTCA software. Cells were seeded directly onto E‐plate 16. Changes in baseline impedance resulting from cell number increase were recorded by the microelectrodes. Proportional changes in impedance were expressed in cell index (CI).

Gao W., Li J.Z., Chen S., Chu C., Chan J.Y, & Wong T. (2017). BEX3 contributes to cisplatin chemoresistance in nasopharyngeal carcinoma. Cancer Medicine, 6(2), 439-451.

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Real-Time Cell Proliferation Monitoring

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Real-Time cell kinetic analyzer xCELLigence RTCA (ACEA Biosciences). was used to monitor the dynamic changes of cell proliferation. Data analysis was performed using RTCA Control Unit and the preinstalled RTCA software. For real-time proliferation assay, E-plate 16 was used. Cells were seeded directly onto E-plate. Changes in baseline impedance resulting from the increase of cell numbers were monitored by gold micro-electrodes located at the bottom of E-plate. The proportional changes in impedance were recorded continuously and expressed as cell index (CI). Change of CI with time was monitored continuously for 72 h.

Gao W., Li J.Z., Chen S.Q., Chu C.Y., Chan J.Y, & Wong T.S. (2016). Decreased brain-expressed X-linked 4 (BEX4) expression promotes growth of oral squamous cell carcinoma. Journal of Experimental & Clinical Cancer Research : CR, 35, 92.

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In vitro T Cell Killing Assay

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In vitro T cell killing was analyzed on an xCELLigence RTCA instrument (ACEA Biosciences). Briefly, tumor cells were added into a xCELLigence E-Plate. The plate was loaded into the instrument to begin data acquisition. Twenty-four hours later, MART-1–enriched T cells were added into the plate, at indicated tumor–T cell ratios, together with MART-1 peptide (500 ng/ml). The cells were harvested 3 days later. T cell killing was determined by the percentage of changes in cell index or impedance in electric current caused by T cells compared with tumor cells cultured alone. The changes in cell index were expressed as changes in area under the curve, which was calculated using with Microsoft Excel.

Tu E., McGlinchey K., Wang J., Martin P., Ching S.L., Floc’h N., Kurasawa J., Starrett J.H., Lazdun Y., Wetzel L., Nuttall B., Ng F.S., Coffman K.T., Smith P.D., Politi K., Cooper Z.A, & Streicher K. (2022). Anti–PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR-mutated NSCLC. JCI Insight, 7(3), e142843.

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Real-Time Cytotoxicity Assay for CAR T Cells

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Cytotoxicity assays were run on an xCelligence RTCA (real-time cell analysis) instrument (ACEA Biosciences) according to the manufacturer's instructions. Briefly, 1 × 10⁴ 3T3-mCD19 target cells were plated per well on an E-Plate 96. The next day, CAR T cells were resuspended in fresh complete medium without IL2 and added onto target cells at 10:1, 5:1, or 1:6 E:T ratios, and growth was monitored up to 172 hours.

Boucher J.C., Li G., Kotani H., Cabral M.L., Morrissey D., Lee S.B., Spitler K., Beatty N.J., Cervantes E.V., Shrestha B., Yu B., Kazi A., Wang X., Sebti S.M, & Davila M.L. (2020). CD28 Costimulatory Domain–Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function. Cancer immunology research, 9(1), 62-74.

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Real-Time Cell Proliferation Assay

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The RTCA impedance assay (xCELLigence RTCA, ACEA Biosciences) is a noninvasive real-time monitoring of cell proliferation, cell size and morphology, continuously providing data over days. Three separate 16-well plates with electrodes embedded are controlled and monitored in parallel. The instrument is placed in a standard CO₂ cell culture incubator and is powered and controlled via a cable connected to the control unit housed outside the incubator. The functional unit of a cellular impedance assay is a set of gold microelectrodes fused to the bottom surface of a microtiter plate well (E-Plates®). The impedance magnitude is dependent on the number of cells, the size and shape of the cells, and the cell-substrate attachment quality and is reported as a Cell Index parameter. Cells are seeded at a concentration of 5000 cells per well in culture medium. 5-FU is added 48 hours after cells seeding, by removal of culture medium and replacing by 5-FU diluted in culture medium at concentrations of 1, 2, 5, 10, 25, 50 and 100 μM (200 μL per well). Cell proliferation was monitored during 50 hours.

Virgone-Carlotta A., Lemasson M., Mertani H.C., Diaz J.J., Monnier S., Dehoux T., Delanoë-Ayari H., Rivière C, & Rieu J.P. (2017). In-depth phenotypic characterization of multicellular tumor spheroids: Effects of 5-Fluorouracil. PLoS ONE, 12(11), e0188100.

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Kinetic Analysis of Tumor Cell Lysis

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Kinetic analysis of tumor cell lysis was performed using iCELLigence and xCELLigence RTCA (ACEA Biosciences, San Diego, CA, USA). Twenty thousand tumor cells were seeded onto E-Plate L8 PET (ACEA Biosciences). After 24-h culture, T cells or control media were added. The cell index (CI) was recorded every 1 h, and cytotoxicity was calculated as {CI (control medium)−CI (effector cells)}/CI (control medium).
To analyze the additional effect of adding G47Δ, the tumor cells were first infected with G47Δ after 24 h of culture, and the supernatant was replaced with fresh culture media after 1 h of infection. After another 1 h culture, T cells or control media were added, and CI was recorded every hour.

Chalise L., Kato A., Ohno M., Maeda S., Yamamichi A., Kuramitsu S., Shiina S., Takahashi H., Ozone S., Yamaguchi J., Kato Y., Rockenbach Y., Natsume A, & Todo T. (2022). Efficacy of cancer-specific anti-podoplanin CAR-T cells and oncolytic herpes virus G47Δ combination therapy against glioblastoma. Molecular Therapy Oncolytics, 26, 265-274.

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Real-Time Cytotoxicity Assay Using xCELLigence

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The xCELLigence® RTCA (ACEA Biosciences) system provides real-time, label-free and non-invasive monitoring of cell health by electrical impedance. This system uses microplates coated with gold electrodes (E-plates) on the bottom of each well. The impedance of current flow in each single well is measured as unitless parameter called cell index (CI) and depends on size, shape and number of adherent cells attaching to the surface of each well bottom. Upon cell death, CI decreases as a result of rounding and detaching of cells from the surface. Neuroblastoma cells were cultivated in E-plates (15.000 cells/well). After 48 h, target cells were incubated with effector cells and antibodies for up to 72 h. For calculations CI was normalized to the time of effector cell addition (NCI). Specific lysis was calculated according to the formula: ((NCI targets only-NCI test)/NCI targets only) × 100%.

Arendt A.M., Heubach F., Maier C.P., Giardino S., Jung G., Kowalewski E., Rabsteyn A., Amorelli G., Seitz C., Schlegel P., Handgretinger R, & Lang P. (2023). Targeting GD2 after allogeneic SCT: effector cell composition defines the optimal use of ch14.18 and the bispecific antibody construct NG-CU (GD2-CD3). Cancer Immunology, Immunotherapy, 72(11), 3813-3824.

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Real-time monitoring of HUVEC invasion

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These assays were performed using E-16-well plates and the xCELLigence RTCA technology (Acea Bioscience) as described [12 (link)]. Microelectrodes placed on the bottom of plates, detect impedance changes which are proportional to the number of adherent cells. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value. HUVECs (1x10⁴ cells/well) suspended in growth medium, were seeded in E-16-well plates and allowed to grow for ~24 hours until they form a confluent monolayer, prior to seeding THP-1 cells (1x10⁴ cells/well) in growth medium plus/minus 10 nM [SRSRY]. When HUVECs are challenged with invading cells, there is a drop in electrical resistance within 2–10 hours which is monitored in real-time as the Cell Index changes due to invasion of the endothelial monolayer. The experiments were performed twice in quadruplicate.

Yousif A.M., Minopoli M., Bifulco K., Ingangi V., Di Carluccio G., Merlino F., Motti M.L., Grieco P, & Carriero M.V. (2015). Cyclization of the Urokinase Receptor-Derived Ser-Arg-Ser-Arg-Tyr Peptide Generates a Potent Inhibitor of Trans-Endothelial Migration of Monocytes. PLoS ONE, 10(5), e0126172.

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Real-Time Cell Growth and Clonality Assays

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After PC cells were seeded into 16-well plates, xCELLigence RTCA (ACEA Biosciences) was performed to examine cell growth ability to acquire real-time data for 96 h uninterruptedly. For plate clonality assays, a total of 1,000 PC cells with or without a coculture system were seeded into each well of a 6-well plate and maintained in medium at 37°C and 5% CO₂ for 2 weeks. Then, the number and size of colonies in each group were counted after the cells were stained with crystal violet (Beyotime). Cell proliferation was also measured by EdU assay (RiboBio) according to its specification in 96-well plates.

Sun Z., Sun D., Feng Y., Zhang B., Sun P., Zhou B., Du L., Wang Y., Fan Z., Yang J., Li Y., Hu S, & Zhan H. (2021). Exosomal linc-ROR mediates crosstalk between cancer cells and adipocytes to promote tumor growth in pancreatic cancer. Molecular Therapy. Nucleic Acids, 26, 253-268.

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