Protocols for alamarBlue®

alamarBlue® protocol:

QC test for alamarBlue®

This quality control (QC) method can be used to determine the standard absorbance values for alamarBlue® under selected experimental conditions. This control is designed to be used for investigations into how values are affected by type of plate, and the chosen media.


Materials and equipment:

  • 0.1 M potassium phosphate buffer, pH7.4
  • 10 ml test tube
  • Pipette capable of accurately dispensing 0.4 ml
  • Plate reader with the filters 570 nm and 600 nm (Alternative filters can be used – see bottom of page)
  • Dynatech flat bottom plate

Procedure:

  1. Shake alamarBlue® to mix before use
  2. Pipette 0.4 ml of alamarBlue® into a test tube
  3. Dilute to 10 ml with phosphate buffer
  4. Mix well
  5. Pipette 100 μl into each well of a clear, flat bottom microplate
  6. Read absorbance at appropriate wavelengths
Wave length (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)

Expected results for quality control (QC) test – using Dynatech flat bottomed plates

N.B. Absorbance values may be affected by the type of plate (whether round or flat bottom) and the plate manufacturer (See Appendix 4 in the technical datasheet for further information about the effect of plates on absorbance).

No QC protocol is recommended for fluorescence since fluorescence units are arbitrary and the scale used varies widely from one instrument to another.

For an indication of the absorbance and fluorescence values for the fully oxidized and reduced forms of alamarBlue with different media, please visit Frequently Asked Questions.


Alternative wavelengths

Although it is preferable to use the recommended wavelengths, a correction factor can be calculated allowing the use of one alternative filter.

Recommended Alternative
570 540
600 630

570nm and 600nm are the recommended wavelengths; only one wavelength may be altered for its respective alternative wavelength.