**2.2 Neutral red**

Neutral red assay was first described by Finter in 1969 as a chemosensitivity assay [22, 23]. This assay provides information on cell machinery and quantifies cell viability and measures cell replication. In addition to hepatocytes and erythrocytes, this assay can also be done in non-adherent cells such as fibroblasts. Cells with biotransformation capacity are recommended to be used when neutral red is used to assess the cytotoxicity of chemicals requiring metabolic activation to toxic metabolites [1, 24].

Its principle is based on the ability of viable cells to absorb neutral red, a weak cationic dye, which penetrates the cell membrane and concentrates in the lysosomes of the cell [24]. The mechanism by which the dye penetrates the cell membrane and accumulates in the lysosomes is not well understood. Micropinocytosis with subsequent fusion of vesicles with secondary lysosome was first suggested however entry by non-ionic passive diffusion has always been postulated. Once inside the lysosome, it binds to the anionic and/or phosphate groups of the lysosomal matrix by electrostatic hydrophobic bonds [24–26]. The dye is then extracted from viable cells using an acidified ethanol solution, and the absorbance of the solubilised dye is quantified using a spectrophotometer at 540 nm wavelength.

The uptake of neutral red by viable cells depends on their capacity to maintain pH gradients through the production of ATP [1, 8]. The dye presents a net charge close to zero at physiological pH that enables it to penetrate the cell membrane. Once inside the lysosome, the dye becomes charged and is retained inside the lysosome due to a proton gradient in the lysosome that maintains a pH lower than that of the cytoplasm [1, 8]. As a result, the amount of retained dye is proportional to the number of viable cells (**Figure 2**).

However, alterations in cell surface or lysosomal membranes can modify the uptake of neutral red by viable cells. For example, a variety of chemicals or pharmaceutical agents induce damage to cell surface or lysosomal membrane that may alter or decrease dye uptake and subsequent retention [27]. Due to specific lysosomal capacities in different cells for taking up the dye, neutral red can be used to differentiate between viable, damaged, or dead cells. Viable cells have intact lysosome and tend to absorb more neutral red dye more than dead cells or cells undergoing apoptosis.

*Trypan Blue Exclusion Assay, Neutral Red, Acridine Orange and Propidium Iodide DOI: http://dx.doi.org/10.5772/intechopen.105699*

**Figure 2.** *Cells stained with neutral red (Adapted from Repetto et al. [24]).*

Therefore, uptake of the dye as well as lysosomal integrity are highly sensitive indicators of cell viability. In addition, the results of neutral red assay are dependent on (a) the degree of acute toxicity (b) number of viable cells in the culture which determine the timing of the assay [25, 26, 28].

Neutral red dye is non-specific and non-toxic and is often used as a counterstain for dyes such as trypan blue dye. Therefore, neutral red assay poses some advantages over other cytotoxicity assays as it is very sensitive, simple, cheap, readily quantifiable, presents less interference and does not require equipment or unstable reagents such as tetrazolium salts that measure lactate dehydrogenase enzyme activity by the chemical reduction of the salts to formazans [22, 29, 30]. The neutral red assay also has advantages, which include speed and reproducibility of data. Also, neutral red estimates can be done on the same cell culture alongside protein determination.

Nevertheless, there are limitations of the neutral red assay such as underestimation of the toxicity of chemicals, which require metabolic activation to a toxic product and of substances, which bind to serum proteins [22, 29, 30]. On the contrary, some chemicals may induce irreversible precipitation of the neutral red dye into fine, needle-like crystals, which may result in an overestimation of the toxic effects [27]. Some chemicals have a localised effect on lysosomes, and this may result in low or high uptake thus leading to overestimation or underestimation of cellular toxicity. Therefore, this assay is suitable for detecting chemicals such as chloroquine that selectively target lysosome and alter its pH thus inducing a greater effect of neutral red uptake than most chemicals [24]. This causes an overestimation of the toxic effects induced by these chemicals. Changes in cell proliferation may also interfere with the estimation of lysosomal function [26, 28, 29]. To prevent this, the assay can be performed in conjunction with other cytotoxicity assays. However, performing neutral red estimates followed by determination of enzymatic function such as lactate dehydrogenase and glucose-6-phosphate dehydrogenase or total protein determination in the same cell culture may lead to a reduction in the amount of protein estimated to be present in the cell culture.

### **2.3 Acridine orange**

Acridine orange is a heterocyclic organic compound that was first extracted from coal tar. It acts as a weak basic nucleic acid dye, which can permeate the cell membrane and accumulate in these acidic organelles such as lysosome in a pH dependent manner [31]. Acridine orange is hydrophobic, which enables it to permeate the cell membrane quickly, enter the cytoplasm and accumulate in the lysosome [31–33]. Therefore, acridine orange can be used to stain lysosome, vacuoles, and nucleus where it specifically binds to double stranded DNA and RNA in living cells by intercalation or electrostatic attractions (**Figure 3**). Acridine orange stabilises the pigment-DNA complexes via charge neutralisation of DNA backbone phosphate group [33, 35].

Under acridine orange staining, lysosome fluoresce bright-red or orange red at a wavelength of 590 nm whereas the nucleus and cytoplasm emit green fluorescence at a wavelength of 525 nm. Due to its low molecular weight (256 g/mol), it rapidly diffuses into the cytoplasm of living cells to bind to DNA and RNA [36, 37]. Once bound to single stranded DNA or RNA, red fluorescence is emitted whereas green fluorescence is emitted when bound to double stranded DNA. As a result, acridine orange can be used in apoptotic studies to stain apoptotic cells orange or red depending on the degree of loss of membrane integrity. This dye also has the capacity to label dead cells thus differentiating the apoptotic cells into early apoptosis (green) and late apoptosis (orange red) hence it offers superior accuracy than the older methods [10, 11, 38].

However, fluorescence response of acridine orange is dependent upon the concentration of acridine orange used, the solvent used in dissolving the dye, fixation, time of staining, ions present, ionic strength of the medium, pH, temperature, and complexing substrate [37, 39, 40]. Lowering the concentration of acridine orange causes a decrease in fluorescence whilst an increase in acridine orange concentration will cause a metachromatic shift. In addition, acridine orange is usually co-stained with other

#### **Figure 3.**

*Stromal cell line, HS-5 cells, stained with acridine orange, which stained the nucleus of the cells (Adapted from Okeke [34]).*

#### *Trypan Blue Exclusion Assay, Neutral Red, Acridine Orange and Propidium Iodide DOI: http://dx.doi.org/10.5772/intechopen.105699*

dyes such as ethidium bromide and propidium iodide [10, 37–41]. The stock solution of acridine orange (100 mg/ml) and ethidium bromide/propidium iodide (100 mg/ ml) is usually made in phosphate buffered saline (PBS) or distilled water and stored in a foil-wrapped bottle at 4°C. This is because acridine orange is light-sensitive and can degrade upon exposure to light.

Co-staining of cells with acridine orange and ethidium bromide or propidium iodide provides information on nuclear morphology (perinuclear chromatin condensation, nuclear collapse, and eventual fragmentation) [10, 37, 40, 41]. Ethidium bromide and propidium iodide can bind with core histones of DNA nucleosome structure but lack the metachromatic property of acridine orange and only stain dead cells when combined with acridine orange [10, 37, 40, 41]. This enables earlier identification of damaged cells by suppressing the DNA-specific green fluorescence induced by acridine orange. Ethidium bromide and propidium iodide are impermeable to intact cell membranes and intercalate the DNA and emits red or orange fluorescence when cells lose their membrane integrity [10, 12, 37–40]. In the presence of two dyes, the cells stain red or orange because the molar concentration of propidium iodide/ethidium bromide is more than 100× greater than that of acridine orange thus propidium iodide/acridine orange has greater affinity and specificity for nucleic acids than acridine orange. In addition, the active accumulation of acridine orange in the cells stops upon death, which reduces the concentration of acridine orange in the cells thereby making the red fluorescence of ethidium bromide/propidium iodide is more apparent [12, 38, 40].

Therefore, dual staining of cells with acridine orange and propidium iodide/ ethidium bromide aids the detection of four main types of cells: (a) Viable cells with uniform green nuclei with organised chromatin structure (b) Necrotic cells with uniform orange red or red nuclei with organised structure due to loss of membrane integrity (c) Early apoptotic cells with irregular structured green nucleic but chromatin is condensed as apoptotic bodies or green patches/fragments (d) late apoptotic cells, with orange or red nuclei with extremely condensed or fragmented chromatin [10, 11, 38, 40–42]. Thus, since the cellular detail and nuclear outline are distinct, co-staining cells with acridine orange and ethidium bromide/propidium iodide offers a rapid, stable, sensitive, and easy-to-perform way to simultaneously visualise and identify all the possible nuclear stages with increased accuracy and ease of interpretation. In addition, light microscopy of cells co-stained with these dyes also provide visualisation of a complete morphological profile of an apoptotic cell [12, 37, 41–43]. Therefore, this assay also permits the staining and scoring of multiple specimens in batches thus it is cost-effective and labour saving. It also works well at room temperature and not subject to interference by extracellular enzymes.

However, fluorescence microscope is required to perform this dual staining thus expertise in this field is paramount. In addition, propidium iodide and ethidium bromide are carcinogenic and can cause debilitating effects to the DNA [11, 12, 38]. Interestingly, acridine orange is also carcinogenic and has been used as an antitumour agent in targeting different cancer cells via photosensitization as it selectively binds to malignant cells compared to normal cells [40–42]. Accumulation of acridine orange in the lysosome at low pH is also crucial for photosensitization thus resulting in the release of oxygen radicals, especially in malignant cells.
