Automated Ratio Imaging Using Nuclear-Targeted FRET Probe-Expressing Cells for Apoptosis Detection

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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Ratio imaging of intracellular ion concentration

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100130432 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1160-1161

Author(s):

Stephen R. Bolsover

Keyword(s):

Individual Cell ◽

Video Camera ◽

Field Of View ◽

Ion Concentration ◽

Fluorescence Signal ◽

Ion Imaging ◽

Ratiometric Fluorescence ◽

Ratio Imaging ◽

The Mean ◽

The Many

The field of intracellular ion concentration measurement expanded greatly in the 1980's due primarily to the development by Roger Tsien of ratiometric fluorescence dyes. These dyes have many applications, and in particular they make possible to image ion concentrations: to produce maps of the ion concentration within living cells. Ion imagers comprise a fluorescence microscope, an imaging light detector such as a video camera, and a computer system to process the fluorescence signal and display the map of ion concentration.Ion imaging can be used for two distinct purposes. In the first, the imager looks at a field of cells, measuring the mean ion concentration in each cell of the many in the field of view. One can then, for instance, challenge the cells with an agonist and examine the response of each individual cell. Ion imagers are not necessary for this sort of experiment: one can instead use a system that measures the mean ion concentration in a just one cell at any one time. However, they are very much more convenient.

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Cytotoxic Effect of the Combination of Gemcitabine and Atorvastatin Loaded in Microemulsion on the HCT116 Colon Cancer Cells

International Journal of Pharmaceutical and Clinical Research ◽

10.25258/ijpcr.v9i2.8298 ◽

2017 ◽

Vol 9 (2) ◽

Cited By ~ 1

Author(s):

Mayson H. Alkhatib ◽

Dalal Al-Saedi ◽

Wadiah S. Backer

Keyword(s):

Colon Cancer ◽

Cancer Cells ◽

Anticancer Drugs ◽

Therapeutic Potential ◽

Morphological Changes ◽

In Vitro Study ◽

Colon Cancer Cells ◽

Apoptosis Detection ◽

Hct116 Cells

The combination of anticancer drugs in nanoparticles has great potential as a promising strategy to maximize efficacies by eradicating resistant, reduce the dosage of the drug and minimize toxicities on the normal cells. Gemcitabine (GEM), a nucleoside analogue, and atorvastatin (ATV), a cholesterol lowering agent, have shown anticancer effect with some limitations. The objective of this in vitro study was to evaluate the antitumor activity of the combination therapy of GEM and ATVencapsulated in a microemulsion (ME) formulation in the HCT116 colon cancer cells. The cytotoxicity and efficacy of the formulation were assessed by the 3- (4,5dimethylthiazole-2-yl)-2,5-diphyneltetrazolium bromide (MTT) assay. The mechanism of cell death was examined by observing the morphological changes of treated cells under light microscope, identifying apoptosis by using the ApopNexin apoptosis detection kit, and viewing the morphological changes in the chromatin structure stained with 4′,6-diamidino-2-phenylindole (DAPI) under the inverted fluorescence microscope. It has been found that reducing the concentration of GEM loaded on ME (GEM-ME) from 5μM to 1.67μM by combining it with 3.33μM of ATV in a ME formulation (GEM/2ATV-ME) has preserved the strong cytotoxicity of GEM-ME against HCT116 cells. The current study proved that formulating GEM with ATV in ME has improved the therapeutic potential of GEM and ATV as anticancer drugs.

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Titanium Oxide Nanoparticles Improve the Chemotherapeutic Action of Erlotinib in Liver Cancer Cells

Current Cancer Therapy Reviews ◽

10.2174/1573394715666191204101739 ◽

2020 ◽

Vol 16 (4) ◽

pp. 337-343

Author(s):

Shaimaa E. Abdel-Ghany ◽

Eman El-Sayed ◽

Nour Ashraf ◽

Nada Mokhtar ◽

Amany Alqosaibi ◽

...

Keyword(s):

Liver Cancer ◽

Cancer Cells ◽

Titanium Oxide ◽

Phase Distribution ◽

Oxide Nanoparticles ◽

Titanium Oxide Nanoparticles ◽

Liver Cancer Cells ◽

M Phase ◽

Apoptosis Detection ◽

Novel Method

Background: Hepatocellular carcinoma is the second leading cause of cancer-related deaths among other types of cancer due to lack of effective treatments and late diagnosis. Nanocarriers represent a novel method to deliver chemotherapeutic drugs, enhancing their bioavailability and stability. Methods: In the present study, we loaded gold nanoparticles (AuNPs) and titanium oxide nanoparticles (TiO2NPs) with ERL to investigate the efficiency of the formed composite in inducing apoptosis in HepG2 liver cancer cells. Cytotoxicity was assessed using MTT assay and cell phase distribution was assessed by flow cytometry along with apoptosis detection. Results: Data obtained indicated the efficiency of the formed composite to significantly induce cell death and arrest cell cycle and G2/M phase. IRF4 was downregulated after treatment with loaded ERL. Conclusion: Our data showed that loading ERL on TiO2NPs was more efficient than AuNPs. However, both nanocarriers were efficient compared with control.

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Detecting apoptosis as a clinical endpoint for proof of a clinical principle

Ophthalmologica ◽

10.1159/000513584 ◽

2020 ◽

Author(s):

Piero Zollet ◽

Timothy E.Yap ◽

M Francesca Cordeiro

Keyword(s):

Drug Development ◽

Therapeutic Response ◽

Imaging Techniques ◽

Brain Diseases ◽

Promising Strategy ◽

Sight Loss ◽

Apoptosis Detection ◽

Novel Therapeutic Strategies

The transparent eye media represent a window through which to observe changes occurring in the retina during pathological processes. In contrast to visualising the extent of neurodegenerative damage that has already occurred, imaging an active process such as apoptosis has the potential to report on disease progression and therefore the threat of irreversible functional loss in various eye and brain diseases. Early diagnosis in these conditions is an important unmet clinical need to avoid or delay irreversible sight loss. In this setting, apoptosis detection is a promising strategy with which to diagnose, provide prognosis, and monitor therapeutic response. Additionally, monitoring apoptosis in vitro and in vivo has been shown to be valuable for drug development in order to assess the efficacy of novel therapeutic strategies both in the pre-clinical and clinical setting. Detection of Apoptosing Retinal Cells (DARC) technology is to date the only tool of its kind to have been tested in clinical trials, with other new imaging techniques under investigation in the fields of neuroscience, ophthalmology and drug development. We summarize the transitioning of techniques detecting apoptosis from bench to bedside, along with the future possibilities they encase.

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Apoptosis detection: a purpose-dependent approach selection

Cell Cycle ◽

10.1080/15384101.2021.1919830 ◽

2021 ◽

pp. 1-8

Author(s):

Maojuan Guo ◽

Bin Lu ◽

Jiali Gan ◽

Shuangcui Wang ◽

Xijuan Jiang ◽

...

Keyword(s):

Apoptosis Detection

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Fast-dissolving ocular films of riboflavin acetate conjugate for treatment of keratoconus in UVA-CXL procedure:ex vivopermeation, hemolytic toxicity and apoptosis detection

Expert Opinion on Drug Delivery ◽

10.1517/17425247.2014.873028 ◽

2014 ◽

Vol 11 (3) ◽

pp. 325-343

Author(s):

Akhilesh Tiwari ◽

Neeraj Kumar Gangwar ◽

Kamla Pathak

Keyword(s):

Apoptosis Detection ◽

Hemolytic Toxicity

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Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.3.c747 ◽

1999 ◽

Vol 276 (3) ◽

pp. C747-C757 ◽

Cited By ~ 50

Author(s):

Sujatha Jayaraman ◽

Joachim Biwersi ◽

A. S. Verkman

Keyword(s):

First Generation ◽

Spectral Characteristics ◽

Dual Wavelength ◽

Reduced Form ◽

Excitation Wavelength ◽

Transmembrane Conductance Regulator ◽

Fluorescent Indicators ◽

3T3 Fibroblasts ◽

Ratio Imaging ◽

Ratiometric Measurement

The fluorescence of quinolinium-based Cl− indicators such as 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl− by a collisional mechanism without change in spectral shape. A series of “chimeric” dual-wavelength Cl− indicators were synthesized by conjugating Cl−-sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl−-sensitive moiety [excitation wavelength (λex) 350 nm, emission wavelength (λem) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl−-insensitive moiety because of its different spectral characteristics (λex 380 nm, λem 546 nm), insensitivity to Cl−, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl−-sensitive indicators were α-MQ-α′-dimethyl-AQ-xylene dichloride and trans-1,2-bis(4-[1-α′-MQ-1′-α′-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl− sensitive; Stern-Volmer constants 82 and 98 M−1) and 565 nm (Cl−insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 μM, 10 min). Changes in Cl− concentration in response to Cl−/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl− indicators for ratiometric measurement of Cl− concentration.

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