Fig. 6 Relative fluorescent units (r.f.u.) for different cell concentration of Sf21 insect cells at different times of incubation. While at lower cell lines concentrations an increase of r.f.u. can be observed, higher cell concentrations facilitate further reduction of resorufin to dihydroresorufin which leads to a decreasing signal. Sf 21 cells were incubated at 27 °C and a pH of 6.4. This can also lead to decreasing r.f.u. due to the shift of the resazurin/resorufin equilibrium to resazurin
16
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19
Chapter 2
Assaying Cellular Viability Using the Neutral Red Uptake Assay
Gamze Ates, Tamara Vanhaecke, Vera Rogiers, and Robim M. Rodrigues
Abstract
The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cat- ionic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been intro- duced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.
Key words Viability assay, Neutral red uptake, HepG2
1 Introduction
The neutral red uptake (NRU) assay is a viability assay based on the ability of living cells to incorporate and bind neutral red (NR) [1].
This weak cationic eurhodine dye can penetrate cells by nonionic diffusion at physiological pH. Once NR is in the cell, it accumu- lates intracellularly in lysosomes, where a proton gradient assures a more acidic pH and the dye becomes charged [2]. Xenobiotics can lead to alterations of the cell surface or lysosomal membrane, which results in a decreased uptake and binding of NR. As such, the NRU assay allows to assess membrane permeability and lysosomal activ- ity, making it possible to differentiate viable, damaged, or dead cells. Cytotoxicity is expressed as a concentration-dependent reduction of the uptake of NR after exposure to the xenobiotic, thus providing a sensitive, integrated signal of both cell integrity and cell growth inhibition [1]. The NRU has miscellaneous biologi- cal applications and is commonly used to evaluate the cytotoxicity
Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_2, © Springer Science+Business Media LLC 2017
of a variety of chemical substances such as pharmaceuticals and cosmetics [3, 4]. Several validation studies have been set up for the NRU as a test for cytotoxicity [5]. In 2000 a NRU test on Balb/
c 3T3 mouse fibroblasts to assess phototoxicity, was regulatory accepted in all EU member states and in 2004 it was adopted as an official Organisation for Economic Co-operation and Development (OECD) test guideline (TG 432) [6]. In 2013, the European Commission Joint Research Centre has published a recommenda- tion on the use of the 3T3 NRU assay in which it stresses the valid- ity of the NRU in a weight-of-evidence approach to predict acute oral toxicity of chemicals in a regulatory setting [7]. The facility of the NRU assay permits automation, which improves throughput and allows fast and reliable screening of a large amount of test compounds in a relatively short time span [4, 8].
For the purpose of this book chapter, the NRU is described on HepG2 cells. This human hepatoma cell line originates from a 15-year-old Caucasian male and is widely employed in hepatotox- icity studies. Under proper culture conditions, HepG2 cells display (limited) hepatocyte-like features and are therefore often utilized as an alternative in vitro model for human hepatocytes [9–11].
2 Materials
1. Incubator: 37 ± 1 °C, 90 ± 5% humidity, 5.0 ± 1% CO2/air.