Rtca software

Manufactured by Agilent Technologies

Sourced in United States

The RTCA software is a tool used to monitor and analyze real-time cellular responses in a non-invasive manner. It provides a platform for recording and interpreting data from Agilent's Real-Time Cell Analyzer (RTCA) instruments.

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48 protocols using rtca software

Quantifying Invasive Potential of PANC-1 Cells

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The invasive activity of PANC-1 cells was determined with xCELLigence^® technology (ACEA Biosciences, San Diego, supplied by OLS, Bremen, Germany) as outlined in detail in previous publications [39 (link),40 (link)]. The lower side of the CIM plate-16 porous membrane was coated with a 1:1 mixture (v/v) of collagen I and collagen IV (30 μL) to facilitate adherence of the cells and thus enhance the duration of signal recording. Prior to cell seeding, the surface of the upper chamber was covered with a thin monolayer of 5% (v/v) growth factor-reduced Matrigel (BD Biosciences, Heidelberg, Germany) diluted 1:20 with basal medium, as detailed elsewhere [72 (link)]. After the Matrigel solification, each well was loaded with 60,000 or 80,000 cells in standard growth medium (see above). Cells were allowed to settle in the laminar flow hood for 30 min at RT, after which the assay was started and run for 24–48 h. Data acquisition (with signal recording every 15 min) and analysis was performed with the RTCA software (version 1.2, ACEA).

Ungefroren H., Thürling I., Färber B., Kowalke T., Fischer T., De Assis L.V., Braun R., Castven D., Oster H., Konukiewitz B., Wellner U.F., Lehnert H, & Marquardt J.U. (2022). The Quasimesenchymal Pancreatic Ductal Epithelial Cell Line PANC-1—A Useful Model to Study Clonal Heterogeneity and EMT Subtype Shifting. Cancers, 14(9), 2057.

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Real-time Cell Migration and Invasion Assay

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Migration and invasion assays were performed in 16-well CIM plates in an xCELLigence RTCA DP system (ACEA Biosciences) using matrigel basement membrane matrix (BD) for real-time cell migration analysis as described previously.²² (link) In brief, 30,000 cells per well were seeded as 5 duplicates in serum-free medium at the upper compartment of the CIM plates coated with or without matrigel. Serum-complemented medium was added to the lower compartment of the chamber, and then we started measurement in xCELLigence RTCA DP system and analyzed the CI (cell index) curves to determine cell invasion activity. For negative controls, we added serum-free medium at both upper and bottom chambers. The cell index representing the amount of migrated cells was calculated with the RTCA Software from ACEA Biosciences. At least three independent experiments were carried out; for each independent experiment, five duplicates were performed for each group.

Cheng C., Zhou Y., Li H., Xiong T., Li S., Bi Y., Kong P., Wang F., Cui H., Li Y., Fang X., Yan T., Li Y., Wang J., Yang B., Zhang L., Jia Z., Song B., Hu X., Yang J., Qiu H., Zhang G., Liu J., Xu E., Shi R., Zhang Y., Liu H., He C., Zhao Z., Qian Y., Rong R., Han Z., Zhang Y., Luo W., Wang J., Peng S., Yang X., Li X., Li L., Fang H., Liu X., Ma L., Chen Y., Guo S., Chen X., Xi Y., Li G., Liang J., Yang X., Guo J., Jia J., Li Q., Cheng X., Zhan Q, & Cui Y. (2016). Whole-Genome Sequencing Reveals Diverse Models of Structural Variations in Esophageal Squamous Cell Carcinoma. American Journal of Human Genetics, 98(2), 256-274.

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NK Cell Cytotoxicity Assay with Engineered Endothelial Cells

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Mouse NK cells were isolated from fresh BALB/c or C57BL/6 spleens 18 h after poly I:C injection (100 μg intraperitoneally). After red cell lysis, NK cells were purified with MagniSort Mouse NK cell Enrichment Kit (Invitrogen), followed by CD49b MACS-sorting (Miltenyi). This cell population was highly selected for NK cells with a purity of >9%. Human NK cells from PBMCs were purchased from StemCell Technologies containing >99% NK cells.
NK cell killing assays were performed on the XCelligence SP platform (ACEA BioSciences). 96-well E-plates (ACEA BioSciences) were coated with collagen (Sigma-Aldrich) and 4 × 10⁵ WT, B2m^−/−Ciita^−/−, or B2m^−/−Ciita^−/− Cd47 tg miECs or WT, B2M^−/−CIITA^−/− or B2M^−/−CIITA^−/− CD47 tg hiECs were plated in 100 μl cell-specific media containing 1 ng ml⁻¹ mouse or human IL-2 (Peprotech). After the Cell Index value reached 0.7, NK cells were added with an effector cell / target cell (E/T) ratio of 0.5/1, 0.8/1 or 1/1. As a negative control, cell treated with 2% Triton X100 was used. Some wells were pretreated with mouse Cd47 or human CD47-blocking antibody (BioXCell) with 10 μg ml⁻¹ media for 2 h. Data were standardized and analyzed with the RTCA software (ACEA).

Deuse T., Hu X., Gravina A., Wang D., Tediashvili G., De C., Thayer W.O., Wahl A., Garcia J.V., Reichenspurner H., Davis M.M., Lanier L.L, & Schrepfer S. (2019). Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nature biotechnology, 37(3), 252-258.

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NK and Macrophage Cell Killing Assays

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NK cell killing assays and macrophage killing assays were performed on the XCelligence SP platform and MP platform (ACEA Biosciences). Special 96-well E-plates (ACEA Biosciences) were coated with collagen (Sigma-Aldrich), and 4 × 10⁵ WT, B2M⁻^/⁻CIITA⁻^/⁻, B2M⁻^/⁻CIITA⁻^/⁻ CD47 tg, and B2M⁻^/⁻CIITA⁻^/⁻ rhCD47 tg hiECs were plated in 100 µl cell-specific medium. After the cell index value reached 0.7, rhesus NK cells or rhesus macrophages were added with an E:T ratio of 0.5:1, 0.8:1, or 1:1 with or without 1 ng/ml rhesus IL-2 (MyBioSource). As a negative control, cells were treated with 2% Triton X-100 in cell-specific media (data not shown). Data were standardized and analyzed with the RTCA software (ACEA Biosciences).

Deuse T., Hu X., Agbor-Enoh S., Jang M.K., Alawi M., Saygi C., Gravina A., Tediashvili G., Nguyen V.Q., Liu Y., Valantine H., Lanier L.L, & Schrepfer S. (2021). The SIRPα–CD47 immune checkpoint in NK cells. The Journal of Experimental Medicine, 218(3), e20200839.

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Real-Time Cell Growth Analysis

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xCELLigence RTCA SP Station and Analyzer (ACEA Biosciences, San Diego, CA, USA) was used for real-time cell growth analysis. The 1000–5000 U87MG cells per well were seeded, and impedance of the wells were measured with 20 min intervals for 24 or 202 h. The amount of cell growth was analyzed and plotted using the RTCA Software (ACEA Biosciences).

Kurt I.C., Sur I., Kaya E., Cingoz A., Kazancioglu S., Kahya Z., Toparlak O.D., Senbabaoglu F., Kaya Z., Ozyerli E., Karahüseyinoglu S., Lack N.A., Gümüs Z.H., Onder T.T, & Bagci-Onder T. (2017). KDM2B, an H3K36-specific demethylase, regulates apoptotic response of GBM cells to TRAIL. Cell Death & Disease, 8(6), e2897-.

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Real-Time Cell Proliferation and Migration Assay

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We seeded 5 × 10⁴/mL cells in E-plate for proliferation analysis and CIM plate for migration analysis, respectively. The CIM plate contains the upper chamber and the lower chamber. We added 30 μL serum-free medium into the upper chamber. We added 165 μL serum medium into the lower chamber. The parameters of RTCA DPlus instrument were set (ACEA Biosciences, Hangzhou, China). Then, cell samples were put in the instrument, which can automatically record a real-time cellular proliferation or migration curves. Cell index representing the amount of proliferation and migration cells was calculated with RTCA software from ACEA Biosciences (San Diego, CA).

Xu P., Zhang J., Wang M., Liu B., Li R., Li H., Zhai N., Liu W., Lv C, & Song X. (2022). hnRNPL-activated circANKRD42 back-splicing and circANKRD42-mediated crosstalk of mechanical stiffness and biochemical signal in lung fibrosis. Molecular Therapy, 30(6), 2370-2387.

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Real-Time Cell Migration Monitoring

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An xCELLigence Real‐Time Cell Analysis System (RTCA; ACEA Biosciences, CA, USA) in a humidified 37°C 5% CO₂ incubator was used to monitor HCC cell migration. Briefly, the cells (5 × 10⁴/well) were added to the instrument chamber (E‐plate16, ACEA Biosciences), with appropriate treatments being applied. Cells were then monitored over a 20 h period, with electrical impedance being measured with gold microelectrodes to detect proliferation. This impedance was used to calculate the cell index value corresponding to cell proliferation, with this value being calculated every 10 min for the 20 h period with the RTCA software (ACEA Biosciences).

Liu S., Huttad L., He G., He W., Liu C., Cai D., Chen H, & Qiu J. (2022). Long noncoding RNA HULC regulates the NF‐κB pathway and represents a promising prognostic biomarker in liver cancer. Cancer Medicine, 12(4), 5124-5136.

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Real-time Cell Migration and Invasion Assay

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Migration and invasion assays were performed in 16-well CIM plates in an xCELLigence RTCA DP system (ACEA Biosciences) using matrigel basement membrane matrix (BD) for real-time cell migration analysis as described previously [10 (link)]. Briefly, 30,000 cells per well were seeded as 5 duplicates in serum-free medium at the upper compartment of the CIM plates coated with or without matrigel. Serum-complemented medium was added to the lower compartment of the chamber, and then started measurement in xCELLigence RTCA DP system and analyzed the CI (Cell Index) curves to determine cell invasion activity. For negative controls, we added serum-free medium at both upper and bottom chambers. The concentration of matrigel was 1:6 for KYSE150 cells and 1:10 for KYSE140 cells. The cell index representing the amount of migrated cells was calculated with the RTCA Software from ACEA Biosciences (San Diego, CA). At least three independent experiments were carried out; for each independent experiment, 5 duplicates were performed for each group.

Song B., Cui H., Li Y., Cheng C., Yang B., Wang F., Kong P., Li H., Zhang L., Jia Z., Bi Y., Wang J., Zhou Y., Liu J., Wang J., Zhao Z., Zhang Y., Hu X., Shi R., Yang J., Liu H., Yan T., Li Y., Xu E., Qian Y., Xi Y., Guo S., Chen Y., Wang J., Li G., Liang J., Jia J., Chen X., Guo J., Wang T., Zhang Y., Li Q., Wang C., Cheng X., Zhan Q, & Cui Y. (2015). Mutually exclusive mutations in NOTCH1 and PIK3CA associated with clinical prognosis and chemotherapy responses of esophageal squamous cell carcinoma in China. Oncotarget, 7(3), 3599-3613.

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Real-Time Cellular Proliferation and Migration Assay

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A real-time cellular proliferation/migration assay system (ACEA Biosciences, Inc., Hangzhou, China) was applied to analyze the proliferation and migration of L929 cells. The cells were cultured in E-plate (proliferation plate) and CIM-plate (migration plate) in a real-time cellular analysis (RTCA) instrument (RTCA DPlus), and proliferation and migration curves were automatically recorded. The number of proliferating and migrating cells was calculated through RTCA software from ACEA Biosciences (San Diego, CA).

Zhang J., Chen X., Chen H., Li R., Xu P., Lv C., Liu B, & Song X. (2020). Engeletin ameliorates pulmonary fibrosis through endoplasmic reticulum stress depending on lnc949-mediated TGF-β1-Smad2/3 and JNK signalling pathways. Pharmaceutical Biology, 58(1), 1114-1123.

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Panc1 Cell Migration Assay using xCELLigence DP

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The xCELLigence^® DP system (ACEA Biosciences, San Diego, CA, USA, distributed by OLS, Bremen, Germany) was employed for recording random cell migration of Panc1 cells. CIM plates-16 were prepared according to the instruction manual and previous descriptions [3 (link),14 (link)]. The underside of the upper chambers of the CIM plate-16 was coated with 30 μL of collagen I (400 μg/mL). In all assays, RPMI with 1% fetal bovine serum was present in both the upper and lower chambers of each well of a CIM plate-16. The upper chamber of each well was loaded with 50,000–60,000 cells immediately after the addition of 5 ng/mL TGF-β1 to the cell suspensions. Data acquisition was done at intervals of 15, 30, or 60 min and analyzed with RTCA software (ACEA).

Ungefroren H., Otterbein H., Fiedler C., Mihara K., Hollenberg M.D., Gieseler F., Lehnert H, & Witte D. (2019). RAC1B Suppresses TGF-β1-Dependent Cell Migration in Pancreatic Carcinoma Cells through Inhibition of the TGF-β Type I Receptor ALK5. Cancers, 11(5), 691.

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