**2. Experimental dyes used in measuring cytotoxicity**

### **2.1 Trypan blue dye exclusion assay**

Trypan blue dye exclusion assay is one of the most frequently used routine methods to determine cell viability [9, 13]. It involves the selective staining of dead cells with trypan blue and microscopic examination on haemocytometer [9]. It was developed in 1975 to measure viable cells and provide information on cell mortality [2]. Trypan blue, a non-permeable cell membrane dye, is an azo dye derived from toluidine. This is a vital stain used in bioscience to exclusively stain necrotic (dead) cells (**Figure 1**). Therefore, this assay is based on the principle that viable cells have intact cell membranes, which trypan blue cannot penetrate thus, trypan blue is excluded from viable cells [5].

In contrast, trypan blue dye penetrates cell membrane of necrotic (dead) cells due to loss of cell membrane integrity and enters the cytoplasm. Under light microscope, only necrotic (dead) cells absorb this blue colour [15]. The trypan blue staining technique is usually performed on a single sample or relatively small number of samples from simple experiments [6].

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

**Figure 1.** *Cells stained with trypan blue. Neubauer chamber counter (Adapted from Hong et al. [14]).*

However, there have been some questions raised about the integrity of the trypan blue technique. These include:


to a cell with time may influence the expression of *P53* and *P21* genes, which are crucial in cell cycle. *P53* helps in making intracellular protein located in the nucleus and plays a major role in cell cycle; controlling cell division and cell death whereas *P21* is a cell cycle inhibitor including cell cycle arrest, apoptosis, and DNA repair [19–21]. Therefore, the impact of prolonged exposure of trypan blue dye to a cell on *P53* and *P21* genes expression imposes a challenge that contradicts the use of this assay [18].

In conclusion, there is need for optimisation of concentration and incubation time depending on the type of cell involved to eliminate the possibility of this dye causing a gradual damage on the cells. Also, trypan blue dye may likely underestimate cellular damage when performed immediately after treating cells with a cytotoxic agent. As a result, many researchers combine this technique with another dye to ensure the cytotoxic results are robust. Benchtop instruments have also been designed to automate imaging and improve the biased analysis steps of this assay.
