Protocols for alamarBlue®

alamarBlue® protocol:

Calculations for using alamarBlue® in spectrophotometry with different filters

  1. Make up alamarBlue® (AB) as directed in the package insert (1/10 dilution in 100 μI media). 
  2. Measure the absorbance of alamarBlue® in media at the lower wavelength (LW) filter and at the higher wavelength (HW) filter. 
  3. Measure the absorbance of 100 ul media only (blank) at the two wavelengths. 
  4. Subtract the absorbance values of media only from the absorbance values of alamarBlue® in media.  

Where: AOLW = absorbance of oxidized form at the lower wavelength 

 AOHW = absorbance of oxidized form at the higher wavelength 


AOLW = Absorbance of AB in media  -  Absorbance of media only

AOHW = Absorbance of AB in media  -  Absorbance of media only


  1. Calculate correction factor (Ro): 

Correction factor for different filters:

RO = AOLW/ AOHW


  1. Calculate percentage difference in reduction between treated and control cells in cytotoxicity/proliferation assays: 

Percentage difference in reduction (equation 5)

=

ALW - (AHW RO) for test well  


ALW - (AHW RO) for control well

x100

 


Where: ALW = absorbance at lower wavelength minus the media blank 

 AHW = absorbance at higher wavelength minus the media blank 

 Ro = Correction factor (calculated in step 5) 

  1. If the alternative calculation to find the percent reduction of alamarBlue® is required, use the following equation: 

Percentage reduction of alamarBlue®  (equation 6) =   [ ALW - (AHW  x RO) ]  x 100

Example Data:

E. coli was added to Mueller Hinton broth and alamarBlue® to a final concentration of 1 x 106 cfu/ml. The broth was incubated at 36oC and readings taken of 1 ml samples every 30 minutes, absorbance was measured at 560 nm and 605 nm.

  Absorbance 560 nm Absorbance 605 nm
10% alamarBlue® in 100 ul media 0.474 0.815
100 ul media 0.082 0.037
Test well after 60 min incubation 0.639 0.889
Test well after 120 min incubation 0.797 0.893

Table 4: Example data (test wells contain 10% alamarBlue® in 100 ul media containing cells)

Where AOLW  = 0.474 - 0.82   

                         = 0.392

Where AOHW = 0.815 - 0.037 

                   = 0.778

 

             RO = 0.392 / 0.778

                  = 0.504                   

[Using equation 6]

Where: ALW =absorbance at lower wavelength minus the media blank

          AHW =absorbance at higher wavelength minus the media blank

              RO = Correction factor (calculated in previous step)

After 60 minutes incubation:                  After 120 minutes incubation:
ALW = 0.639 - 0.082 = 0.557   ALW
AHW = 0.889 - 0.037 = 0.857   AHW
RO = 0.504   RO
Percentage reduction of alamarBlue®   Percentage reduction of alamarBlue®
= [0.557 - (0.852 x 0.504)] x 100    = [0.715 - (0.856 x 0.504)] x 100
= 13%   =28%