An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity
| Autor: | Natalia V. Kirienko, S. B. Panina, Jingqi Pei |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Cell Survival
High-throughput screening General Chemical Engineering 010402 general chemistry 01 natural sciences Article General Biochemistry Genetics and Molecular Biology Automation chemistry.chemical_compound Cell Line Tumor Rotenone Humans Propidium iodide Cytotoxicity Cell Nucleus Cell Death General Immunology and Microbiology 010405 organic chemistry Differential staining General Neuroscience Reproducibility of Results Resazurin Mitochondria 0104 chemical sciences Staining chemistry Biochemistry Biological Assay Trypan blue Formazan |
| Zdroj: | J Vis Exp |
| ISSN: | 1940-087X |
| DOI: | 10.3791/61295-v |
| Popis: | The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods. |
| Databáze: | OpenAIRE |
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