alamarBlue is a cell viability assay reagent which contains the cell permeable, non-toxic, and weakly fluorescent blue indicator dye resazurin. It has been extensively referenced and used in a wide range of research areas. 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. Popular alamarBlue is a preferred alternative to other cell proliferation assays such as MTT, XTT, 3H Thymidine, or neutral red.
alamarBlue Work Principle
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. alamarBlue is a direct indicator of cell health and it detects the level of oxidation during respiration, quantitatively measuring cell viability and cytotoxicity.
Here are detailed protocols for use of alamarBlue.
Quality Control Testing Method for alamarBlue
Wavelength (nm) | Average Absorbance (Standard Deviation) |
540 | 0.145 (0.002) |
570 | 0.225 (0.003) |
600 | 0.313 (0.004) |
630 | 0.116 (0.002) |
AlamarBlue Cell Viability
Protocol for assaying cell viability with a test compound:
Note: The suggested optimal cell count is 1 x 104 cells/ml. It may varybetween different cell lines.
Note: For positive control well, add 10µl of ultrapure sterile water.
Determine LD50 Using alamarBlue
alamarBlue can be used to determine the median lethal dose LD50 value and similar assays testing the effects of a toxin, radiation, or pathogen on cell cultures. Use semi-log graph paper to plot the percent of untreated control for each dilution of a given test agent on the y-axis vs. the concentration of the test agent on the x-axis. To determine the LD50 endpoint from the graph, read from where the 50% intercepts the dose.
Fig.1 Response curve to the concentration along the x-axis.
Measuring Cytotoxicity or Proliferation Using alamarBlue
Note: The suggested optimal cell count is 1 x 104 cells/ml. It may varybetween different cell lines.
Note: The optimum incubation time may vary between cell types.
Determining Optimum Length of Incubation and Plating Density
The two variables which most affect the response of cells to alamarBlue are length of incubation time and number of cells plated. It is suggested that the plating density and incubation time should be determined for each cell line using the following steps.
Note: It is suggested that the plates remain in incubation overnight and measurements be made the following day at 24 h. Two kinds of information can be obtained from this data:
Percentage reduction of alamarBlue= [ (O2 x A1) - (O1 x A2) ] x 100%/ [ (R1 x N2) - (R2 x N1) ]
Where:
O1 = molar extinction coefficient (E) of oxidized alamarBlue (blue) at 570 nm
O2 = E of oxidized alamarBlue at 600 nm
R1 = E of reduced alamarBlue (red) at 570 nm
R2 = E of reduced alamarBlue at 600 nm
A1 = absorbance of test wells at 570 nm
A2 = absorbance of test wells at 600 nm
N1 = absorbance of negative control well (media plus alamarBlue but no cells) at 570 nm
N2 = absorbance of negative control well (media plus alamarBlue but no cells) at 600 nm
Only one appropriate substitute wavelength may be used.
alamarBlue Spectrophotometry Calculations with Different Filters
Note: AOLW = absorbance of alamarBlue in media - absorbance of media only, lower wavelength
AOHW = absorbance of alamarBlue in media - absorbance of media only, higher wavelength
Percentage difference in reduction= [ (A LW - (AHW x Ro) for test well ] x 100%/ A LW - ( AHW x Ro ) for control well.
ALW = absorbance at lower wavelength minus the media blank
AHW = absorbance at higher wavelength minus the media blank
Ro = correction factor
Percentage reduction of alamarBlue = [A LW- ( AHW x Ro ) ] x 100%
Advantages of Using alamarBlue
Other Protocols