Neutral red uptake assay

Repetto, G., del Peso, A. and Zurita, J.L. (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protocols, 3, 1125-1131. 

Jones, P.A. and King, A.V. (2003) High throughput screening (HTS) for phototoxicity hazard using the in vitro 3T3 neutral red uptake assay. Toxicology In Vitro An International Journal Published in Association with BIBRA, 17, 703-708. 

The discovery of Zanamivir as a potent and selective inhibitor of influenza virus sialidase prompted several researchers to investigate the synthesis and structure-activity relationship studies of Neu5Ac2en-based compounds as potential sialidase inhibitors. Exploration of these SAR studies were undertaken to optimize inhibitory activity and to improve the physicochemical properties of the sialic acid-based influenza virus sialidase inhibitor. A few in vitro assays are commonly employed to measure the effectiveness of influenza virus sialidase inhibitors. The first involves a fluorometric assay that measures release of a synthetic fluorophore following its cleavage from Neu5Ac by sialidase. Dye-uptake assay, such as the Neutral Red uptake assay, measures the uptake of a vital stain, Neutral Red in cell culture. The process requires intact membranes and active metabolism in the cell, and is expressed as percent protective rate against virus infection. The plaque-reduction assay is used to measure sialidase inhibition indirectly in cell culture, and provides some measure of the inhibitor s effect on the viability of the influenza virus. In vitro and in vivo systems for analysis of inhibitors of influenza virus enzymes have been reviewed.71... 

Cytotoxicity—MTT or neutral red uptake assay on mammalian cells... 

Cytotoxicity is the most important parameter to evaluate the biocompatibility of biodegradable polymers in vitro. Various assays are available to measure cell viability after exposure to polymer extracts, such as lactate dehydrogenase leakage assay (LDH), neutral red uptake assay (NRU), and trypan blue assay. Table 20.5 summarizes the different types... 

Cell survival assays Stains survival cells Neutral red uptake assay... 

A variety of mammalian cells have been successfully cultured onto porous silicon surfaces. The first publications on this topic by Bayliss et al. demonstrated that attachment of Chinese hamster ovary (CHO) cells proceeded on porous silicon surfaces to a similar extent as on bulk silicon (Bayliss et al. 1997a, b). This was also confirmed with the neuronal cell line B50 (Bayliss et al. 2000). Cell viability in these studies was determined using two colorimetric assays, the MTT based on enzymatic reduction of a tetrazolium salt to a purple formazan and the neutral red uptake assay. B50 and CHO cells were cultured on bulk silicon, porous silicon, glass, and polycrystalline silicon. Both viability assays suggested that the neuronal cells showed preference for porous silicon above the other surfaces, while CHO cells showed the lowest viability on the porous silicon surface (Bayliss et al. 1999, 2000). The surfaces of the porous silicon used in these early studies were not modified post-etching, and it was not until a study utilized porous silicon surfaces with an oxide layer for cell culture that surface chemistry was found to play a crucial factor (Chin et al. 2001). Rat... 

Assays are frequently needed to detect marked and acute cytotoxicity that may confound the interpretation of cell-based efficacy assays. Neutral red uptake is one of the most commonly used cytotoxicity assays and is used in the regulatory phototoxicity assay on NT3 fibroblasts [13]. It has been show to be more sensitive than assays for mitochondrial reductive capacity such as the tetrazolium reductase assays, ATP depletion assays, or for cell permeabilization or mpture such as dye uptake or lactate dehydrogenase leakage. Lysosomes take up, protonate and trap neutral red when cellular ATP production is sufficient to maintain pH gradients. 

In a study of six mercury compounds, mercury chloride, mercury nitrate, sodium ethylmercurithi-osalicylate, methyl mercury chloride, mercury acetate and phenylmercury acetate in MDCK cells, LLC-PKl cells and human primary proximal tubular cells (hPTC) and non-renal cell lines (SAOS and Hep G2) it was found that all mercury compounds were toxic to all cell types as evidenced by neutral red uptake, thymidine incorporation and the MTT assay [189]. However, sodium ethylmercurithiosalicylate, methyl mercury chloride and phenylmercury acetate were one order of magnitude more toxic than the other compounds. In addition the GSH synthesis inhibitor L-buthionine sulfoximine (BSO) potentiated the toxicity of all mercury compounds [189]. In a study using primary rabbit proximal tubular cells it was also shown that methyl mercury chloride is more toxic than mercury chloride [190]. Differences in the extent and rate of metal uptake were also evident. Maximum cellular uptake of Hg " occurred within 6-24 hr after exposure and was not concentration-dependent, whereas maximum uptake of CHgHg" occurred within 3 hr of exposure and was concentration- dependent [190]. 

Many other cell lines have been used to address specific types of toxicity. Some of these have reached a level of validation that is sufficient for in vitro screening purposes. For example, the 3T3 NRU assay was regarded as an acceptable screen for hazard identification of potential phototoxicity [27,28], This assay utilized the in vitro 3T3 cell line as a generic cell line and Neutral Red uptake as a cytotoxicity measurement. The compound in question is subjected to UV irradiation to assess the effect of UV rays on compound induced toxicity to 3T3 cells. Another example is the use of the MCF-7 cell proliferation assay as an in vitro screen for endocrine disrupters [29],... 

Several intertest comparisons of cytotoxicity assays have been undertaken, Sina et al. (1992) compared the following assay procedures leucine incorporation (a general cytotoxicity test), MTT dye reduction (an indicator of mitochondrial damage), and neutral red uptake. They found that none of the end points accurately predicted in vim eye irritating potential, but the MTT dye reduction method gave the overall best correlation. Christian and Diener (1996) noted that the neutral red uptake a.ssay had merit for ranking potential ocular irritation, particularly the weaker irritants. 

A number of in vitro models have been developed for assessing the phototoxic potential of chemicals. The most widely used in vitro phototoxicity assay is the in vitro 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT). This assay was validated by ECVAM and subsequently adopted by the Organization for Economic Co-operation Development (OECD) as Test Guideline (TG) 432 (OECD,... 

Normal human keratinocyte neutral red uptake (NHK NRU) assay In vitro... 

Under validation (ECVAM, ICCVAM) we have the BALB/c 3T3 neutral red uptake (NRU) cytotoxicity assay and the normal human keratinocyte NRU cytotoxicity assay. Both methods are based on the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR penetrates cell membranes and accumulates in lysosomes. Alterations in the cell surface lead to decreased uptake and binding of NR (Nottingham and Spielmann, 1995 Spielmann et al, 1999). 

Shopsis, C. (1989). Validation study ocular irritancy prediction with the total cell protein, uridine uptake, and neutral red assays applied to human epidermal keratinocytes and mouse 3t3 cells. In Alternative Methods in Toxicology, Vol. 7 (Goldberg, A.M., Ed.). Mary Arm Liebert, New York, pp. 273-287. 

The test is based on an in vitro assay of the uptake of the dye, neutral red (NR), in Balb/c 3T3 fibroblasts. It was developed to detect the phototoxicity induced by the combined interaction of the test substance and light of the wavelength range from 315 to 400 nm, the so-called UVA. The cytotoxicity is evaluated in the presence (+UVA) or absence (-UVA) of UVA light exposure, after application of a nontoxic dose of the compound. The cytotoxicological impact is assessed via the inhibition of the fibroblasts to take up the vital dye NR (NR is a weak cationic dye, penetrating easily into the cell membrane by a nonionic diffusion and accumulates in the lysosomes) one day after the initial treatment. Normally, healthy cells may incorporate and bind NR. Alterations of the cell surface or the lysosomal membranes, however, lead to a decreased uptake and binding of the dye. 

Phospholipidosis (e.g., Nile red, lysotracker dyes, electron microscopy of lysosomal multilamellar bodies), vacuolization, autophagy, lysosomal uptake assays for cell viability (e.g., neutral red)... 

Function of mitochondria is also commonly monitored as an indicator of cellular toxicity. Mitochondrial uptake and retention of the fluorescent dye, rhodamine 123, can be visualized microscopically. Biochemical measurements of mitochondrial function include the ATP-ADP ratio and dehydrogenase activity with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which yields a colored formazan product upon reduction. The dye, neutral red (3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride), targets lysosomes, and its retention is inversely related to cytotoxicity. Commercially available versions of the MTT and neutral red assays have been adapted to microtiter plate formats to provide highly efficient screening assays. Examples of how cell-type-specific functions can be followed as indicators of cell toxicity are included in Table 8.1. 

The most common assay systems consist of cell cultures that are treated with the drug in the culture medium. Toxicity is commonly assessed by release of intracellular enzyme, for example, lactate dehydrogenase or aspartate transaminase, into the culture medium or by other indicators such as decreases in the rate of radiolabeled amino acid or nucteotide precursor incorporation into macromolecules. A decrease in the intracellular uptake of the vital dye, neutral red, has also... 

The antiviral effect of any compound must be measured against its toxicity. Once cells are seeded and treated following the guidelines above, a neutral red dye uptake assay (2) is performed to assess toxicity. Other assays of cytotoxicity (e.g., MTT) can be substituted for the procedure described below. Note that this procedure assesses toxicity under culture and treatment conditions that are identical to those used for the antiviral analyses, thereby permitting a determi-... 

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