MTT

The MTT assay is a colourimetric assay for measuring the metabolic activity of cells.

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a yellow tetrazole, is reduced to a purple formazan dye when mitochondrial reductase enzymes are active in cells. A solubilisation solution is added to dissolve the insoluble purple formazan product into a coloured solution. The absorbance of this coloured solution can be quantified using a spectrophotometer.

Conversion of the MTT is often used as a measure of viable (living) cells. Changes in metabolic activity can take place when changes in cell number occur (via cell death or cell proliferation), or changes in the activity of the reductase enzymes occurs, while the number of viable cells is constant.

When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing cell death/proliferation, or changes in the metabolism of the cells, can be deduced through the production of a dose-response curve.

As potential agents (drug intermediates, formulations etc) may stimulate or inhibit metabolic activity by affecting the viability or performance of the cells, the cytotoxicity of those agents can be determined.

Neutral Red Uptake

The Neutral Red Uptake assay can be used to determine cell viability. Neutral Red (NR), a weak cationic dye, penetrates cellular membranes, enters cells via non-ionic diffusion and accumulates intracelluarly in lysosomes. Viable cells take up the NR dye, damaged or dead cells do not, thus the Neutral Red Uptake (NRU) assay can be used as a direct measure of cell viability (as opposed to MTT which indirectly measures cell viability via metabolic activity).

Cell incorporated NR is released from the cells using a solubilisation solution such as acidified ethanol. The absorbance of the NR can be quantified using a spectrophotometer. Cytotoxicity can be deduced through the production of a dose-response curve after chemical exposure.

Alterations of the cell surface or the lysosomal membrane may lead to lysosomal fragility and other changes that gradually become irreversible. Such changes brought about by the action of external agents may result in a decreased uptake and binding of NR.

A decrease in the uptake of neutral red dye in treated cell cultures following a test chemical exposure is used to determine relative toxicity, distinguishing between viable, damaged, or dead cells.

LDH release

Membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. One commonly measured molecule is lactate dehydrogenase (LDH) present in the cytosol. LDH converts pyruvate to lactate with concomitant interconversion of NADH and NAD+ when oxygen is absent or in short supply.

When cells are damaged, LDH is released into the culture medium. The LDH, released from the cytoplasm of chemically-treated cells into the culture medium, reduces NAD+ to NADH in the presence of lactate. NADH in turn converts a tetrazolium dye (INT) to a red soluble formazan product which can be measured spectrophotometrically. The level of LDH released into the medium is therefore indirectly determined. The LDH levels in culture medium are directly proportional the extent of cell membrane damage.

As the level of LDH activity corresponds with cell number also, this assay can be used to determine cell proliferation via lysing the cells and measuring the LDH stored within the cytosol. The LDH levels in the cell lysate are directly proportional cell growth.