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Cellular and Molecular Toxicology of Nanoparticles
Published in Vladimir Torchilin, Handbook of Materials for Nanomedicine, 2020
A. Zielińska, D. Santos, J. R. Campos, A. Santini, P. Severino, A. A. M. Shimojo, S. B. Souto, E. B. Souto
The cell can die by apoptosis and/or necrosis [58]. Mitochondrial function plays an important role in the onset of apoptosis since it is the release of proteins present in the mitochondrial membrane that initiate the apoptosis mechanism. One of these molecules is cytochrome C, which when released into the cytosol can lead to the activation of caspases 9, 7 and 3—the protease enzymes playing essential roles in apoptosis and inflammation [22, 55]. Thus, the activity of caspases can be used as a biomarker for apoptosis. There are already kits in the market that evaluate the activity of these enzymes. These kits consist of a peptide susceptible of being cleaved by a caspase. The peptide is marked with a probe that emits fluorescence or changes the color [58]. The condensation of chromatin and fragmentation of the DNA occurs during apoptosis. The detention of these processes can be employed for determining the initiation of apoptosis, during in vitro toxicity tests by using DNA-laddering techniques known as the TNUEL assay. The TUNEL assay detects the DNA fragments by binding to the fragmented ends of the DNA. This bond can be detected by tagging [55]. A substantial amount LDH is leaked into the cell medium after cell damage [9]. The LDH is a biomarker of cell death that it is soluble in the culture medium. Its presence indicates the occurrence of cell damage and can be detected by colorimetry. In the market, there are a few of commercial kits for LDH detection [4, 58].
Valve interstitial cells under impact load, a mechanobiology study
Published in Journal of Medical Engineering & Technology, 2023
Dylan Goode, Ruby Dhaliwal, Hadi Mohammadi
To study the effect of impact on VICs, a terminal transferase dUTP nick end-labelling (TUNEL) assay was run on the slides of each leaflet which was divided into 4 × 4 layers. DNA fragmentation resulting from apoptosis is a basis for the TUNEL assay. The TUNEL assay is utilised to distinguish these fragmentations [50]. The existence of nicks in the DNA, which is a basis for the assay, is detected by terminal transferase. Terminal transferase is an enzyme that catalyses the accumulation of dUTPs that are labelled with a marker [50,51]. In addition, we counterstained the slides using propidium iodide, which is a known marker that can segregate living cells from dead cells [51]. This marker is a fluorescent stain that pervades impaired cellular membranes and interrelates with nuclear DNA. In a cellular fluorescent image, a non-apoptotic cell is detected blue (nuclei) while an apoptotic cell shows green (nuclei). In this study, both staining techniques were used on each slide to increase accuracy of detecting damaged and undamaged cells which allows for assessment of positively stained cells twice. A confocal microscope (Zeiss LSM® Imaging Software, Zeiss Co., Jena, Germany) was used for imaging each leaflet sample. Each sample has two matching slides (four slices). Each slice was imaged in five arbitrary zones producing an N-value of 60 (three leaflets, four partitions and five images). Due to the existence or lack of nuclei fluorescence within the cell, damaged and non-damaged cells were assessed. The number of green fluorescing cells represented apoptotic cells and the number of the blue (or blue/green) fluorescing cells represented normal cells. The death rate was defined based on the ratio between the number of dead cells (apoptotic) to all cells.