Neutral red assay

Note Inclusion of serum is usually required to facilitate adherence to the plastic 

Resuspend cells of actively growing culture by standard procedure. 

Count cells and accurately allocate appropriate number suspended in medium, calculated to reach about 60-70% confluence at time of addition of test agent. Cell number will vary depending on cell size and growth rate approximate ranges are 9 x 10 to 4 x 10 cells per well. 

Seed 0.2 ml containing desired number of cells to each well of 96-well plates and incubate at 37°C for 24 h, or longer, to achieve desired density. 

Remove medium and add fresh medium containing graded dilutions of test agent. Incubate for desired length of time. At least eight wells should serve as controls and contain medium without xenobiotics. 

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Consider, for example, the neutral red assay developed by Borenfreund and Peumer (1984, 1987). This test has been evaluated with a number of surfactants and... 

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. 

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. 

Babich H, Borenfreund E. Cytotoxic effects of food additives and pharmaceuticals on cells in culture as determined with the neutral red assay. / Pharm Sci 1990 79 592-594. 

Babich H Borenfreund E (1992) Neutral red assay for toxicology in vitro. In Watson RR (ed.) In Vitro Methods in Toxicology, Chapter 17, pp. 237-251. 

Triglia D, Braa S, Yonan C Naughton G (1991) In vitro toxicity of various classes of test agents using the neutral red assay on a human three-dimensional physiologic skin model. In Vitro Cell and Developmental Biology 21 A 239-244. 

Notes Human SH-SY5Y neuroblastoma cells were Incubated for 1 h with different concentrations of each organophosphate (OP). Cytotoxicity was measured by the neutral red assay. Results represent the IC50 values for inhibition of AChE and NTE activities and for cytotoxicity. DBVP = 0,0-dibutyl 0-(2,2-dlchlorovlnyl) phosphate DOVP = 0,0-dloctyl 0-(2,2-dlchlorovlnyl) phosphate. A high ratio of NTE IC50/AChE IC50 Indicates that the OP is likely to cause acute rather than delayed neurotoxic effects in vivo. 

The most commonly used approaches are the neutral red assay (cell viability and membrane damage), the Lowry (labeled proline), Coomassie blue, and Kenacid blue assays (cell proliferation and total cell protein), the MTT or tetrazolium assay (mitochondrial function), and the intracellular lactate dehydrogenase activity test (cell lysis). 

Magwood, S. and S. George. In vitro alternatives to whole animal testing. Comparative cytotoxicity studies of divalent metals in established cell lines derived from tropical and temperate water fish species in a neutral red assay. Mar. Environ. Res. 42 37-40, 1996. 

In comparison to trivalent arsenic, trivalent antimony proved to be five times less cytotoxic in the neutral red assay and one... 

To study cell viability, several probes are proposed, as indicated in Table 3. Two examples will be given in this section. They correspond to either membrane damage (calcein-AM) or disturbance of mitochondrial potential (Alamar blue). Cell viability is often estimated by the Neutral red assay which is widely used in classical spectrometry and, more recently, in fluorimetry because of its increased sensitivity. More often than not, the fluorogenic dye calcein... 

By the neutral red assay in vitro in HepG2, it was possible to evaluate the effects of the PAHs over the cell viability (Babich, 1988). The eytotoxic potential was induced by B[a]P, benzo[k]fluoranthene, benzo[b]fluoranthene, chrysene, benzo[a]anthracene, pyrene, phen-anthrene, fluoranthene but, however, the fluorene, anthracene, acenaphthene and acenaphthylene were not cytotoxic for the same cells. 

One of the most popular bioassay for interferons is termed the cytopathic effect inhibition assay . This assay is based upon the ability of many interferons to render animal cells resistant to viral attack. It entails incubation of the interferon preparation with cells sensitive to destruction by a specific virus. That virus is then subsequently added, and the percentage of cells that survive thereafter is proportional to the levels of interferon present in the assay sample. Viable cells can assimilate certain dyes, such as neutral red. Addition of the dye followed by spectrophotometric quantitation of the amount of dye assimilated can thus be used to quantitate percentage cell survival. This type of assay can be scaled down to run in a single well of a microtitre plate. This facilitates automated assay of large numbers of samples with relative ease. 

Scott-Fordsmand, J. J., Weeks, J. M. and Hopkin, S. P. (2000). Importance of contamination history for understanding toxicity of copper to earthworm Eisenia fetica (Oligochaeta annelida) using neutral-red retention assay, Environ. Toxicol. Chem., 19, 1774-1780. 

Arechabala, B., Coiffard, C., Rivalland, P., Coiffard, L. J. M. and de Roeck-Holtzhauer, Y. (1999). Comparison of cytotoxicity of various surfactants tested on normal human fibroblast cultures using the neutral red test, MTT assay and LDH, J. Appl. Toxicol., 19, 163-165. 

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. 

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. 

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)... 

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... 

Determined on the basis of loss of cell membrane permeability in response to a deliberate modification in culture conditions. Cell viability can be determined by either neutral red or fluorescein diacetate retention assays. Volume 1(14,15). 

Both the neutral red and SRB staining methods present the advantages that they avoid some potential for artifacts related to chemical interference with the tetrazolium and resazurin assay chemistries used to measure cell metabolism and the signals are stable. The obvious and major disadvantage of... 

Cavanaugh, P.F. et al. 1990. A semi-automated neutral red based chemosensitivity assay for drug screening. Invest. New Drugs 8, 347-354. 

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... 

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