Trypan Blue Exclusion Assay, Neutral Red, Acridine Orange and Propidium Iodide

Cytotoxicity and cell viability assessments are very important parameters that are widely used in fundamental research and drug development to determine the safety profile of toxic compounds. These assays measure the degree to which a substance can cause toxic damage to cells or cell death. There are different assays that have been employed to determine the cytotoxicity of substances. These assays either determine enzymatic function, cell viability, mitochondrial activity, lipid metabolism, cell proliferation and/or cell death. These assays entail use of different kinds of dyes such as trypan blue exclusion dye, neutral red, acridine orange and propidium iodide to stain the cells. Trypan blue dye permeates compromised cell membrane to stain necrotic cells. However, this can lead to false positive and false negative results as it does not provide information on sub-lethal injury. As a result, neutral red and acridine orange can be used as counterstains for trypan blue to stain the lysosome of live cells. Acridine orange can also be used to stain nucleic acids in living cells and is usually co-stained with propidium iodide or ethidium bromide. This is because propidium iodide and ethidium bromide permeate only compromised plasma membrane thus co-staining cells with these dyes can provide vital information that can be used to differentiate between live and dead cells.