Determination of the dose–depth distribution of proton beam using resazurin assay in vitro and diode laser-induced fluorescence detection

Kim, Min Jung; Pal, Sukdeb; Tak, Yu Kyung; Lee, Kyeong-Hee; Yang, Tae-Keun; Lee, Su‐Jae; Song, Joon Myong

doi:10.1016/j.aca.2007.05.009

Cited by 15 publications

(10 citation statements)

References 43 publications

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“…doi:10.1371/journal.pntd.0002471.g005 metabolic capacity of these cells to reduce resazurin (also known as Alamar blue) to the highly fluorescent resorufin. It has been shown that there is a positive linear correlation between metabolic reduction of resazurin and the number of viable cells [33,66]. In viable eukaryotic cells, the conversion of resazurin to resorufin is facilitated by mitochondrial reductases and diaphorases such as dihydrolipoamine dehydrogenase (EC 1.8.1.4,), NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) and flavin reductase (EC 1.6.99.1) found in the mitochondria or cytoplasm [33,67].…”

Section: Discussionmentioning

confidence: 99%

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

et al. 2013

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Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.

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Section: Discussionmentioning

confidence: 99%

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

et al. 2013

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“…This molecule is known to react with hydrogen peroxide in combination with HRP enzyme and is commonly used to detect oxidant species in microbial systems (Castaño et al 2018) or under stress conditions (Lefrancois et al 2016;Kovacik and Babula 2017). This dye and derivatives were also used as chemical dosimeter in proton irradiation (Kim et al 2007). Its chemical mechanism towards one-electron oxidation, i.e.…”

Section: Experimental Physico-chemical Approach Of Radiosensitizationmentioning

confidence: 99%

Importance of radiolytic reactions during high-LET irradiation modalities: LET effect, role of O2 and radiosensitization by nanoparticles

et al. 2019

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Background It is striking how chemists and biologists showed an interest for ionizing radiations very soon after their discovery by Roentgen and Becquerel, and initiated parallel developments of these new fields of research: radiation chemistry and radiobiology. Radiation chemistry deals with the chemical effects produced when materials are exposed to ionizing radiations and begun long before 1942 when it was formally named by Burton (Magee 1988). Early pioneering works were achieved by Curie and Debierne in 1901 by observing gas bubbling out of radium salt solutions, Giesel in 1902 (Giesel 1902), Ramsay in 1903, and the progress and enthusiasm persisted throughout the beginning of 20th century (Debierne 1914; Kernbaum 1909

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“…HEK cells have already been used in pharmacological, endocrinological, and toxicological studies and also in other reporter gene studies (Sung et al, 2009;Lodeiro et al, 2009;Lau et al, 2009;Bora et al, 2008;Mao et al, 2007). HEK cells have also been used in radiobiological studies as a biological model for dose-depth distribution of a proton beam using the resazurin assay (Kim et al, 2007) and for analysis of cell cycle checkpoints after irradiation (Yu et al, 2001). In our previous studies with HEK cells, the LET dependence of cellular killing by heavy ions was comparable to that observed in other cell types (Hellweg et al, 2011).…”

Section: Discussionmentioning

confidence: 54%

Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology

Chishti

Hellweg

Berger

et al. 2015

Life Sciences in Space Research

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The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

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Determination of the dose–depth distribution of proton beam using resazurin assay in vitro and diode laser-induced fluorescence detection

Cited by 15 publications

References 43 publications

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Importance of radiolytic reactions during high-LET irradiation modalities: LET effect, role of O2 and radiosensitization by nanoparticles

Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology

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