Redox principle for alamarBlue®

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alamarBlue^® gives a measure of cell proliferation, by detecting the level of oxidation during respiration.

The alamarBlue^® indicator is ideally set up to detect oxidation by the whole of the electron transport chain. As can be seen from Table 1, the midpoint potential of alamarBlue^® is greater than that of any of the other components of the electron transport chain. alamarBlue^® will be reduced by FMNH2, FADH2, NADH, NADPH and cytochromes, since their midpoint potentials are lower than that for alamarBlue^®. Therefore, alamarBlue® is an excellent detector of reduction of all the elements of the electron transport chain.

Table 1: Oxidation reduction potentials in the electron transport system and alamarBlue^®. The midpoint redox potential (Eo’) values were determined at pH 7.0, at 25°C.

alamarBlue^® is an excellent redox indicator for cell proliferation studies because it does not interfere in any way with the reactions of the electron transport chain. This is because the midpoint potential of alamarBlue^® is greater than any of the components of the electron transport chain. Other redox indicators, such as MTT, interrupt certain reactions in the electron transport chain (see below). With alamarBlue^® the cells remain fully functional and healthy, unaffected by the presence of the indicator - a property unique to alamarBlue^®.

Midpoint potentials

The midpoint potential (Eo’) for any half reaction can be defined as the voltage at which equilibrium is obtained between the concentration of oxidized and reduced products. This midpoint potential gives a measure of the strength of an electron donor. For example, in an equation, a component with a more negative midpoint potential is a stronger electron donor, making it a weaker electron acceptor. Example midpoint potentials are given in Table 1. If any two half equations are combined, the midpoint potentials can be used to determine in which direction the electrons will flow.

Since alamarBlue^® has a midpoint potential greater than the midpoint potential of the cytochromes, it can detect oxidation by all the components of the electron transport chain, and it does not interfere with any of the redox reactions of the electron transport chain.

An alternative redox indicator is tetrazolium salt (MTT). MTT has a midpoint potential of -110mV (see Table 1), which enables MTT to be reduced by the electron donors FMNH2, FADH2, NADH and NADPH. However, since the midpoint potential of MTT is intermediate between that of the electron donors and cytochromes, MTT will not be reduced by cytochromes.

Furthermore, if MTT is reduced by FMNH2, FADH2, NADH or NADPH, the electrons released by these donors will not be passed to the cytochromes as would normally happen in the electron transport chain. This shuts down the respiratory chain.