what is alamarBlue?

What is alamarBlue®

alamarBlue® is a cell viability assay reagent which contains the cell permeable, non-toxic and weakly fluorescent blue indicator dye called resazurin. This is a trusted and established reagent which has been available since 1993. alamarBlue® is a useful non-toxic alternative to the commonly used MTT cell viability assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (O’Brien et al. 2000). 

alamarBlue® quantitatively measures proliferation in human, animal, bacterial, fungal and mycobacterial cells. It is useful for cytokine bioassays, cell viability assays and in vitro cytotoxicity determinations as well as cell growth monitoring. 

how to use alamarblue cell proliferation assay

How does alamarBlue® work?

Resazurin is used as an oxidation-reduction (REDOX) indicator that undergoes colorimetric change in response to cellular metabolic reduction. The reduced form resorufin is pink and highly fluorescent, and the intensity of fluorescence produced is proportional to the number of living cells respiring. Through detecting the level of oxidation during respiration alamarBlue® acts as a direct indicator to quantitatively measure cell viability and cytotoxicity.  

Our how to use alamarBlue® page will provide you with useful methods and calculations for you to plan your experiments.

How alamarBlue® acts as a redox indicator

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.


Eo' (mV)

pH7.0 25oC

NAD+ 2H+ + 2e- <----> NADH + H+ -320
NADP+ 2H+ + 2e- <----> NADPH + H+ -320
FAD+ 2H+ + 2e- <----> FADH2 -220
FMN+ 2H+ + 2e- <----> FMNH2 -210
MTTOX1 + 2H+ + 2e- <----> MTTRED -110
cytochromesOX1 +1 e- <----> cytochromesRED +80 to +290
alamarBlue®OX + 2H+ + 2e- <----> alamarBlue®RED 380
O2 + 4H+ + 4e- <----> 2H2O 820

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.

The alamarBlue® cell viability 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 cytochromes, allowing it to detect oxidation by all the components of the electron transport chain. alamarBlue® can be reduced by FMNH2, FADH2, NADH, NADPH and cytochromes, since their midpoint potentials are lower than that for alamarBlue® . This shows that alamarBlue® is an excellent redox indicator for cell proliferation and viability studies because it does not interfere in any way with the reactions of the electron transport chain. With alamarBlue® the cells remain fully functional, viable and healthy, unaffected by the presence of the indicator, which is a property unique to alamarBlue®.

How MTT interrupts the electron transport chain

An alternative redox indicator is tetrazolium salt (MTT), which has a midpoint potential of -110mV (see Table 1). This 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 cannot be reduced by the cytochromes.

The chain is broken when MTT is reduced because the electrons released by the donors will not be passed to the cytochromes as would normally happen in the electron transport chain. This stops the production from the electron transport chain which inhibits respiration, leading to cell death.

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alamarBlue® is manufactured for Bio-Rad by Trek Diagnostic System. U.S. patent 5,501,959