Abstract
Critical cytotoxicity evaluation of pharmaceuticals is necessary for the clinical practice of chemotherapy. To quantitatively evaluate cell viability, currently there are two main types of sensitive methods including real-time cell analysis (RTCA) and CCK-8 assay, in which RTCA records electrochemical signal changes around an incubated cell, whereas CCK-8 is based on the colorimetric method. Despite the different detection principles adopted for the cytotoxicity assessment, the comparison of the two methods in terms of the application scope is lacking. In order to compare and determine the best experimental method for the study of the toxicity of chlorogenic acid extract from taraxacum officinale on dairy cow mammary epithelial cells. The real time cell analysis (RTCA) and CCK-8 method were used to analyze the cytotoxicity of chlorogenic acid extract to BMEC and calculate its IC50. The results of the real time cell analysis method and the CCK-8 method showed that different concentrations of chlorogenic acid extract reduced the viability of dairy cow mammary epithelial cells, and the decrease was most obvious at 400 ug/mL. The IC50 of the two analysis methods were 326.8 and 320.4 ug/mL, respectively. In contrast, the CCK-8 method had limitations in fixed-point determination. However, the real time cell analysis method can monitor the dynamic biological response process of cell growth and proliferation in real time. Therefore, the real time cell analysis method can observe cell growth more intuitively and accurately, compensate for the shortcomings of the CCK-8 method, and it is a new experimental method for studying cytotoxicity.
Highlights
Taraxacum Officinale, commonly called dandelion is herbaceous perennial belongs to the family of Asteraceae, which can be used as a medicinal material or as food
cell counting kit-8 (CCK-8) assay for cell viability In the CCK-8 assay using BMEC cells, different concentrations of CGA extract were prepared for the cytotoxicity evaluation
real-time cell analysis (RTCA) assay for cell viability In RTCA, CGA extract of difffferent concentrations at 12.5, 25, 50, 100, and 200 μg/mL were chosen for recording the cell growth curves, and the IC50 value of 326.8 μg/mL was obtained, which is close to the result of the CCK-8 assay
Summary
Taraxacum Officinale, commonly called dandelion is herbaceous perennial belongs to the family of Asteraceae, which can be used as a medicinal material or as food. Taraxacum Officinale mainly contains various medicinal ingredients such as chlorogenic acid, caffeic acid, total flavonoids, alkaloids, polysaccharides, etc (Ling et al, 1998). Chlorogenic acid is a kind of depsiptic acid belongs to phenolic compound, which can scavenge free radicals in the body and has significant antibacterial (Lou et al, 2011), anti-inflammatory (Liang & Kitts, 2015), antioxidant (Rui et al, 2017), and anticarcinogenic (Yan et al, 2017) activities. According to the analyzed signals, cell-based assays, in principle, can be classified into colorimetric assays (Fischer et al, 2003), luminogenic assays (Elisia et al, 2008), electrochemical methods (Zhou et al, 2018), cell counting methods (Braun et al, 2018), and so on. Two conventional assays are usually applied for in vitro cellular cytotoxicity evaluation because of their easy operation and standardized readout: electrochemical methods as typified by the real-time cell analysis (RTCA) and cell counting kit-8 (CCK-8) assay, respectively
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