Table 4.

Absorbance-based and colorimetric cell viability and cytotoxicity assays ¹.

Cell Viability and Cytotoxicity Assays Absorbance-Based or Colorimetric
		Principal	Advantage	Disadvantage	References
Dye exclusion assays	Trypan blue	membrane integrity	simple, easy to use, economic, fast	hemocytometer required, counting error, difficult to process large number of samples, cannot be distinguished healthy cells and cell function lost alive cells, toxic on mammalian cells	Jonston 2010 [69], Strober 2001 [70], Aslantürk and Celik 2013 [71], Stone et al., 2009 [72], Yip and Auersperg 1972 [73], Ruben 1988 [74]
	Eosin (Erythrosin B)	membrane integrity	economic, versatility, biosafety	time-consuming, labor-intensive, sensitive for the contamination, filling rate, and inter-user variation	Kim et al., 2016 [75], Marmion 1979 [76]
Colorymetric assays	MTT	enzyme (mitochondrial) activity	easy to use, safe, high reproductibility, widly used	organic solvents required, significant well-to-well error, interaction with compounds results false positive, or false negative data	Stone et al., 2009 [72], Aslantürk et al. [77], Bopp and Lettieri 2008 [78], Mosmann 1983 [79]
	MTS		easy to use, rapid, sensitive, economic, specific, inexpensive	measured absorbance level is influenced by incubation time, cell type, and cell number; optimal incubation time: 1–3 h	Berg et al., 1994 [80], Tominaga et al., 1999 [81], Rotter et al., 1993 [82], Buttke et al., 1993 [83], Promega Technical Bulletin [84], Cory et al., 1991 [85], Riss et al., 1992 [86]
	XTT		results water soluble crystals, quick, sensitive, easy-to-use, safe; highly sensitive and accurate	strongly depends on reductive capacity of viable cells due to pH, cellular ion concentration, cell cycle variation, environmental factors	Scudiero et al.1988 [87]
	WST-1		easy to use, high reproducibility, wildly used, no interference with phenol red, water soluble dye, no additional incubation time	relatively long incubation time (2 h)	Ishiyama et al., 1993 [88]
	WST-8		not cell permeable; cells can be used after the assay; water soluble formazan	intracellular metabolic activity can influence the reduction of WST-8	Tominaga et al., 1999 [81], Strober 2001 [70]
	LDH (lactate dehydrogenase)	enzyme (lactate dehydrogenase) activity	reliability; quick, simple evaluation, indicator of cell death	serum incompetence	Decker and Lohmann-Matthes 1988 [89], Schins et al., 2002 [90], Fotakis et al., 2006 [91], Lappalanien et al., 1994 [92]
	SRB (sulforhodamine B)	SRB-protein interaction	simple, fast, sensitive, good linearity with cell number, metabolism independent, high reproducibility	homogenous cell suspension is required; cellular clumps and aggregates interfere with SRB	Skehan et al., 1990 [93]
	NRU (neutral red uptake)	neutral red uptake and lysosomal accumulation	good marker for lysosomal damage, fast and simple evaluation	influenced by pollutants	Borenfreund and Puerner 1984 [94], Repetto et al., 2008 [95], Ringwood et al.1998 [96]
	Crystal violet	binding for proteins and DNA of viable cells	quick; chemical inhibtors can be incorporated into the assay	metabolism affected compounds can not be tested, not able to measure cell proliferation rate	Feoktisova et al., 2016 [66], Geserick et al., 2009 [97], Degterev et al., 2008 [98], Sun et al., 2012 [99], Feoktisova et al., 2011 [100]

¹ Trypan Blue: Staining with trypan blue is one of the oldest viability assays. In a viable cell, the intact membrane will prevent trypan blue from entering cells. In dead or dying cells, trypan blue will enter the cell, staining it blue. This method was traditionally quantified manually using microscopes and hemocytometers, making it very labor-intensive. However, the recent availability of affordable automated cell counters makes this assay less time consuming and more accurate. MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) is a tetrazolium salt that gets reduced by both mitochondrial and extra-mitochondrial dehydrogenases to form insoluble blue formazan crystals, meaning a solubilization step is required before the assay can be read. MTS/XTT: MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) substrates are similar to MTT. However, one advantage is that the reactions are carried out intracellularly in the presence of the intermediate electron acceptor phenazine methosulfate (PMS), which enhances their sensitivity. In addition, the reduced formazan product is soluble and gets released to the culture media, removing the need for the extra solubility step that is required with MTT. However, phenol red in cell culture media, fatty acids, and serum albumin have all been reported to distort data obtained from MTS, XTT, and WST assays over prolonged incubation periods. WST: Water-soluble tetrazolium salts (WSTs) are cell-impermeable tetrazolium dyes that get reduced extracellularly via plasma membrane electron transport, and combined with the electron acceptor PMS to generate water-soluble formazan dyes. LDH assay: Lactate dehydrogenase is a ubiquitous, stable cytoplasmic enzyme that converts lactate to pyruvate. If the cell membrane has been damaged, LDH, and therefore, its enzymatic activity is released from cells and can be detected in cell culture media.