iCELLigence real-time cell analysis system for examining the cytotoxicity of drugs to cancer cell lines

Şener, Leyla Türker; Albeniz, Gürcan; Dinç, Bircan; Albeniz, Işıl

doi:10.3892/etm.2017.4781

Cited by 62 publications

(30 citation statements)

References 28 publications

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“…Each sample was tested in duplicate. In addition, the cells were cultured in quadruplicate with 40 ng/ml IL-17, 1 µM shikonin or both for 96.8 h. The cell index (CI), a measure of the number of cells, was monitored in real-time to assess proliferation using RTCA software ( 37 ). Each assay was repeated three times.…”

Section: Methodsmentioning

confidence: 99%

Shikonin inhibits CEBPD downregulation in IL‑17‑treated HaCaT cells and in an imiquimod‑induced psoriasis model

Lan

Wang

et al. 2020

Mol Med Rep

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Psoriasis is a chronic inflammatory skin disease characterized by well-defined scaly papules and plaques. Interleukin (IL)-17 is involved in its pathogenesis and promotes the proliferation of epidermal keratinocytes through signal transducer and activator of transcription 3 (STAT3) activation. Shikonin, a natural naphthoquinone isolated from Lithospermum erythrorhizon , possesses anti-inflammatory and immunosuppressive properties and can suppress IL-17-induced vascular endothelial growth factor expression by inhibiting the JAK/STAT3 pathway. In the present study, MTS, iCELLigence and RT-qPCR were used to determine the optimal concentration and duration of IL-17 or shikonin acting on HaCaT cells. The changes in the expression levels of genes associated with the IL-6/STAT3 pathway in differentially treated cells were analyzed via RT 2 Profiler™ PCR Array. Small interfering RNA was used to silence the expression levels of the target gene CCAAT/enhancer-binding protein δ (CEBPD). Western blotting and immunohistochemistry were used to evaluate the effect of shikonin on imiquimod-induced psoriasis in mice and the expression levels of CEBPD. Shikonin reversed IL-17-mediated downregulation of the tumor suppressor CEBPD in HaCaT cells. Moreover, low levels of CEBPD in the imiquimod-induced mouse model of psoriasis were restored by shikonin treatment, which ameliorated excessive keratinocyte proliferation. Taken together, these findings suggest that CEBPD plays a key role in the pathogenesis of psoriasis and can be targeted by shikonin as a potential therapeutic strategy.

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Section: Methodsmentioning

confidence: 99%

Shikonin inhibits CEBPD downregulation in IL‑17‑treated HaCaT cells and in an imiquimod‑induced psoriasis model

Lan

Wang

et al. 2020

Mol Med Rep

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“…Live cell assay methods are highly desired because they potentially enable the detailed kinetic analysis of different cytotoxic pathways. Label-free methods utilizing electrical impedance (real-time cell analysis, RTCA), for instance, are able to monitor cytostasis and cytotoxicity in real time yet lack the ability to distinguish the different modes of cell death [ 10 ]. Substantial improvements in traditional microscopy methods, utilizing traditional and/or novel probes (e.g., pSIVA) paired with integrated optical collection, allow for retrospective analysis of image data by algorithms based on morphology and fluorescence parameters [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

A real-time, bioluminescent annexin V assay for the assessment of apoptosis

et al. 2018

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Apoptosis is an important and necessary cell death program which promotes homeostasis and organismal survival. When dysregulated, however, it can lead to a myriad of pathologies from neurodegenerative diseases to cancer. Apoptosis is therefore the subject of intense study aimed at dissecting its pathways and molecular mechanisms. Although many assay methods exist for confirming whether an apoptotic response has occurred in vitro, most methods are destructive and involve laborious operator effort or specialized instrumentation. Here we describe a real-time, no-wash, microplate method which utilizes recombinant annexin V fusion proteins containing evolved binary subunits of NanoBiT™ luciferase. The fusion proteins, a time-released enzymatic substrate, a necrosis detection dye and exogenous calcium ions are delivered via an optimized and physiologically inert reagent directly to cells in culture at the time of treatment or dosing. Luminescent signals proportional to phosphatidylserine (PS) exposure and fluorescent signals generated as a result of loss of membrane integrity are then collected using a standard multimode plate reader at scheduled intervals over the exposure. The resulting luminescent and fluorescent data are then used to define the kinetics and magnitude of an apoptotic response. This study details our efforts to develop, characterize, and demonstrate the features of the assay by providing relevant examples from diverse cell models for programmed cell death.

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“…This technology permits real‐time monitoring of cellular proliferation by recording changes in electrical impedance in an electrode placed on the bottom of the e‐plate wells (Unterweger et al, ). The use of RT‐CES for analyzing the viability of cells exposed to nanoparticles is gaining more attention, as this technique allows for the detection of cell proliferation in real time without the use of dyes and offering more sensitivity and specificity compared with dye‐based assays (Turker Sener, Albeniz, Dinc, & Albeniz, ). In a previous report (Unterweger et al, ), PC‐3 cells were treated with different concentrations of 4.5 nm USPION (0.53, 2.7, 13.4 or 23.7 μg/mL), uncoated and coated with cisplatin.…”

Section: Discussionmentioning

confidence: 99%

Cytotoxicity, cellular uptake and apoptotic responses in human coronary artery endothelial cells exposed to ultrasmall superparamagnetic iron oxide nanoparticles

Palacios-Hernández

Diaz-Diestra

Nguyen

et al. 2020

J of Applied Toxicology

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Ultrasmall superparamagnetic iron oxide nanoparticles (USPION) possess reactive surfaces, are metabolized and exhibit unique magnetic properties. These properties are desirable for designing novel theranostic biomedical products; however, toxicity mechanisms of USPION are not completely elucidated. The goal of this study was to investigate cell interactions (uptake and cytotoxicity) of USPION using human coronary artery endothelial cells as a vascular cell model. Polyvinylpirrolidone-coated USPION were characterized: average diameter 17 nm (transmission electron microscopy [TEM]), average hydrodynamic diameter 44 nm (dynamic light scattering) and zeta potential −38.75 mV. Cells were exposed to 0 (control), 25, 50, 100 or 200 μg/mL USPION. Concentration-and time-dependent cytotoxicity were observed after 3-6 hours through 24 hours of exposure using Alamar Blue and Real-Time Cell Electronic Sensing assays. Cell uptake was evaluated by imaging using live-dead confocal microscopy, actin and nuclear fluorescent staining, and TEM. Phase-contrast, confocal microscopy, and TEM imaging showed significant USPION internalization as early as 3 hours after exposure to 25 μg/mL. TEM imaging demonstrated particle internalization in secondary lysosomes with perinuclear localization. Three orthogonal assays were conducted to assess apoptosis. TUNEL staining demonstrated a marked increase in fragmented DNA, a response pathognomonic of apoptosis, after a 4hour exposure. Cells subjected to agarose gel electrophoresis exhibited degraded DNA 3 hours after exposure. Caspase-3/7 activity increased after a 3-hour exposure. USPION uptake resulted in cytotoxicity involving apoptosis and these results contribute to further mechanistic understanding of the USPION toxicity in vitro in cardiovascular endothelial cells. K E Y W O R D S apoptosis, cytotoxicity, overendocytosis, ultrasmall superparamagnetic iron oxide nanoparticles Abbreviation: USPION, ultrasmall superparamagnetic iron oxide nanoparticles.

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iCELLigence real-time cell analysis system for examining the cytotoxicity of drugs to cancer cell lines

Cited by 62 publications

References 28 publications

Shikonin inhibits CEBPD downregulation in IL‑17‑treated HaCaT cells and in an imiquimod‑induced psoriasis model

Shikonin inhibits CEBPD downregulation in IL‑17‑treated HaCaT cells and in an imiquimod‑induced psoriasis model

A real-time, bioluminescent annexin V assay for the assessment of apoptosis

Cytotoxicity, cellular uptake and apoptotic responses in human coronary artery endothelial cells exposed to ultrasmall superparamagnetic iron oxide nanoparticles

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