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A Simplified Colorimetric Method for Rapid Detection of Cell Viability and Toxicity in Adherent Cell Culture Systems

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A Simplified Colorimetric Method for Rapid Detection of Cell Viability and Toxicity in Adherent Cell Culture Systems JBUON 2018; 23(5): 1505-1513 ISSN: 1107-0625, online ISSN: 2241-6293 • www.jbuon.com E-mail: [email protected] ORIGINAL ARTICLE A simplified colorimetric method for rapid detection of cell viability and toxicity in adherent cell culture systems Widyan Ahmed Alamoudi1*, Faraz Ahmad2*, Sadananda Acharya2, Shafiul Haque3, Khaldoon Alsamman4, Hatem K. Herzallah2, Sultan T. Al-Otaibi2 1Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia-31441; 2Department of Public Health, College of Public Health, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Ara- bia-31441; 3Research and Scientific Studies Unit, College of Nursing & Allied Health Sciences, Jazan University, Jazan, Saudi Arabia; 4Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia-31441. *These authors contributed equally to this work. Summary Purpose: Evaluation of cell viability and toxicity in adher- safranin formed from extraneously added JG-B provides a ent culture systems is of critical relevance for a wide range proficient indicator of cellular health and viability. of disciplines of biomedical sciences research, including can- Results: Using MCF-7, a breast cancer cell line, we provide cer research, toxicology, pharmacology, cell biology, neurol- a proof of concept for the suggested assay and compare it ogy and nanomedicine. Several well-established cytotoxicity with the MTT assay. assays are widely used by researchers, including the most- preferred MTT assay. Nevertheless, there are problems as- Conclusions: Unlike the MTT assay, our JG-B assay does sociated with them, for example; in terms of the time-factor not require a solubilization/extraction step, and hence fol- and solubilization of the formzan crystals before its spec- lows a much simpler and time-efficient protocol suitable troscopic quantification. In this study, we propose a simple, for high-throughput analysis of cell viability in anchorage- fast and cost-effective colorimetric assay that is free of these dependent cell culture models. Additionally, the JG-B cell issues. viability assay reported here can be suitably applied either independently or in complementation with other assays for Methods: Our assay was based upon reductive splitting of the analysis of cellular viability and toxicity in both ana- blue-green colored supravital safranin derivative dye Janus lytic and therapeutic aspects of research. green B (JG-B) to pink colored diethyl safranin by oxidore- ductases of the electron transport chain (ETC) of actively re- spiring mitochondria. Because this conversion can be easily Key words: cancer cells, cisplatin, cytotoxicity, Janus green and reliably followed spectroscopically, measure of diethyl B, mitochondria, MTT Introduction The analysis of cell viability in adherent cul- are available and some are currently being used ture systems is routinely performed in cell cul- for the assessment of cell viability. Colorimeter ture laboratories because of its indispensability is the method of choice for rapid and economi- in the assessment of cell viability and toxicity in cal assessment of cell viability, and several dyes response to extracellular activators, toxic stressors and protocols are currently used for this purpose. and potential pharmaceuticals. Several protocols These include neutral red that binds to lysosomes Correspondence to: Faraz Ahmad, PhD. Department of Public Health, College of Public Health, Imam Abdulrahman Bin Faisal University, Dammam-31441, Saudi Arabia. Tel: +966 553893464, E-mail: [email protected]. Received: 02/02/2018; Accepted; 01/03/2018 1506 Rapid and simple colorimetric cell viability assay [1], propidium iodide that binds to the DNA of mum at 550 nm) [20-23]. Hence, the formation of the damaged cells [2], crystal violet that binds to diethyl safranin can be directly measured spectro- the proteins and DNA [3], Alamar blue (resazurin; photometrically without any requirement of its Al-Nasiry et al. [4]) and Janus green B that binds extraction or elution. to mitochondria [5]. Nevertheless, tetrazolium The uptake and accumulation of JG-B by ac- salts like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- tively respiring mitochondria with a negative nyltetrazolium bromide (MTT) are currently the membrane potential in adherent cells has been most widely employed agents for colorimetric as- employed previously to analyze the cell numbers say of cell viability [6,7]. by some research groups [5,24-27], although the However, several types of agents have been assay is time-consuming and requires error prone shown to reduce MTT leading to erroneous re- steps of ethanol-fixation, washing and de-staining sults in cell viability assays. These include serum preceding spectroscopic analysis of JG-B at 595 albumin [8], metal ions [9,10], plant extracts and nm [5,26]. Moreover, the analysis of cellular up- antioxidants [11,12], vitamins [13] pH of medium take of JG-B as reported previously [5,26] could [14,15] and concentration of glucose, NADH and lead to erroneous results because JG-B can non- NADPH [16]. Nevertheless, many of these con- specifically bind to nuclei and dead cells as well straints can be dealt by extensively washing the as non-mitochondrial cellular compartments [28]. cultured cells before the initiation of exposure to On the other hand, the JG-B cell viability assay MTT [17]. Hence, there is a critical requirement for proposed in this study employs the principle of the employment of complementary cell viability reductive splitting of JG-B into diethyl safranin assay for the validation of the results and infer- and involves the measurement of the absorbance ences obtained from the well-established cell vi- ratio at 550 nm/595 nm, circumventing both the ability assay. de-staining (previously used JG-B assays) [5,26] In addition to the above stated limitations, the and solubilization steps (MTT assay) [17,18]. This MTT assay also requires solubilization/elution of is a major advantage when considering the robust- formazan (the water-insoluble reduction prod- ness, simplicity and time consumption factors. uct) before the spectroscopic analysis [17]. This Employing the principle of reductive splitting makes MTT assay tedious and time-consuming of JG-B into diethyl safranin by actively respiring and, in addition, potentially error prone [18]. In mitochondria in view, we propose a simple and this regard, tetrazolium salts like 2,3-bis-(2-meth- rapid colorimetric assay for the assessment of the oxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-car- viability of the cells in anchorage-dependent cul- boxanilide (XTT), which are designed to produce ture systems. In this study, we provide a proof of water-soluble formazan are suitable alternatives. principle of our JG-B cell viability assay and its However, the employment of XTT in cell viability comparison with MTT assay, providing evidence assessment has been limited because of the net for its suitability as a time-efficient, high through- negative charge of XTT accountable for its largely put and robust assay for the analysis of cell vi- cell impermeable nature [6]. ability/integrity and toxicity, both independently To bypass the time-consuming solubiliza- and in complementation of the established cell vi- tion/extraction step central to MTT cell viability ability protocols based on MTT and neutral red. assay, we have designed a cheap, time-efficient and high-throughput assay for the quantitative analysis of cell viability and toxicity. Briefly, the Methods assay protocol employs 8-[[4-(dimethylamino) Chemicals and reagents phenyl]diazenyl]-N,N-diethyl-10-phenylphenazin- 10-ium-2-amine chloride, commonly referred to All the chemicals/biochemicals used in this study as Janus green B (JG-B). JG-B is a supravital li- were of analytical grade and procured from Merck (Darmstadt, Germany) or Sigma-Aldrich (Steinheim, pophilic cationic mono-azo safranin derivative Germany). Janus green B and MTT were procured from and like other lipophilic cationic dyes including Merck and Sigma, respectively. MTT, JG-B is taken up only by active mitochondria with an intact negative inside membrane potential Mitochondrial reduction of JG-B [19]. Unlike MTT which is reduced by mitochon- In order to assess the reduction of JG-B by the iso- drial dehydrogenases from a yellow colored dye lated mitochondria, 10 μM JG-B with varying amounts to water-insoluble blue formazan product, JG-B is of highly enriched mitochondrial preparations or post- reductively split from a deep greenish-blue color mitochondrial supernatant (PMS) were obtained from dye (absorption maximum at 595 nm) to pink color rat brain cortices (Supplementary methods). Absorbance water-soluble diethyl safranin (absorption maxi- was recorded at 550 and 595 nm before and 10 min after JBUON 2018; 23(5): 1506 Rapid and simple colorimetric cell viability assay 1507 the addition of mitochondria or PMS and the formation Statistics of diethyl safranin was measured as a ratio of absorb- Statistical comparisons between the multiple ance at 550 nm/595 nm. All the experiments involving groups was done by one-way analysis of variance (ANO- animals were carried out in accordance with the institu- VA) followed by post-hoc tests with Dunnett’s correc- tional guidelines for animal care and use in scientific re- tion. Linear or least square non-linear regression analy- search and after approval from the Institutional Review sis of the data was performed using GraphPad Prism 5 Board (IRB), Imam Abdulrahman Bin Faisal University, software program (GraphPad Software, Inc.). The graphi- Dammam, Saudi Arabia. cal representation of data was also done using GraphPad Cell culture Prism 5 software. MCF-7 breast cancer cells were maintained at a sub- confluent state in DMEM medium containing 10% fetal Results calf serum (FCS), 200 μg/ml streptomycin, 200 U/ml penicillin in humidified conditions (95/5% air/CO2) at Absorption spectra of JG-B and its derivative diethyl 37ºC. A well-known anti-cancer drug, cisplatin, was used safranin for inducing cell death in MCF-7 cells.
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