General method for measuring cytotoxicity or proliferation using alamarBlue®

alamarBlue® protocol:

General method for measuring cytotoxicity or proliferation using alamarBlue®

  1. Harvest cells which are in the log phase of growth and determine cell count. Adjust the cell count to1x104 cells/ml (suggested cell density). The optimum cell density may vary between cell types. 
  2. Plate cells and expose to test agent as determined by researcher.  For determining the effect of a test agent on cell growth, ensure correct controls are included e.g. stimulated vs. unstimulated cells.  
  3. Mix by shaking and then aseptically add alamarBlue in an amount equal to 10% of the volume in the well. 
  4. Incubate cultures with alamarBlue for 4 - 8 hours. N.B. The optimum incubation time may vary between cell types. 
  5. Measure cytotoxicity or proliferation using spectrophotometry of fluorescence.

Method for measuring cytotoxicity or proliferation using alamarBlue® by spectrophotometry

  1. Harvest cells which are in the log phase of growth and determine cell count. Adjust the cell count to 1x104cells/ml (suggested cell density). The optimum cell density may vary between cell types. 
  2. Plate cells and expose to test agent as determined by researcher.  For determining the effect of a test agent on cell growth, ensure correct controls are included e.g. stimulated vs. unstimulated cells. 
  3. Mix by shaking and then aseptically add alamarBlue in an amount equal to 10% of the volume in the well. 
  4. Incubate cultures with alamarBlue for 4 - 8 hours. N.B. The optimum incubation time may vary between cell types. 
  5. Measure cytotoxicity or proliferation using spectrophotometry of fluorescence. 
  6. Measure absorbance at wavelengths of 570 nm and 600 nm after required incubation. Use a blank of media only. 

To calculate the percent difference in reduction between treated and control cells in cytotoxicity and proliferation assays (Equation 3): 


Percentage difference between treated and control cells (equation 3)

=

(O2 x A1) - (O1 x A2)


(O2 x P1) - (O1 x P2)

x100


Where: 

  • O1 = molar extinction coefficient (E) of oxidized alamarBlue® (Blue) at 570 nm*
  • O2= E of oxidized alamarBlue® at 600 nm*
  • A1 = absorbance of test wells at 570 nm 
  • A2 = absorbance of test wells at 600 nm
  • P1 = absorbance of positive growth control well (cells plus alamarBlue® but no test agent) at 570 nm 
  • P2 = absorbance of positive growth control well (cells plus alamarBlue® but no test agent at 600 nm

* Only one appropriate substitute wavelength may be used. 


Example data: 

  •  O1 = 570 nm O2 = 600 nm
  •  O1 = 80586 (See table in General method for oxidation coefficients)
  • O2 = 117216 
  •  A1 = 0.65 Observed absorbance reading for test well 
  •  A2 = 0.36 Observed absorbance reading for test well 
  •  P1 = 0.78 Observed absorbance reading for positive control well 
  •  P2 = 0.19 Observed absorbance reading for positive control well

Percentage difference in reduction
 =
(117216 x 0.65) - (80586 x 0.36)

(117216 x 0.78) - (80586 x 0.19)
x 100
 
=
76190 - 29011

 91428 - 15311
x 100
 
=
47179 

76117
 
 
=
62%
 

This would indicate that the amount of reduction in the test well is only 62% of that in the control well, or put another way, that growth in the test well is inhibited by 38% when compared to that of the control.

Wavelength Reduced (R) Oxidized (O)
540nm 104395 47619
570nm 155677 80586
600nm 14652 117216
630nm 5494 34798

Molar extinction coefficients for alamarBlue® at different wavelengths

Alternatively, it may be useful to calculate the percent reduction of alamarBlue® (Equation 1): 


Percentage reduction of alamarBlue® (equation 1)

=

(O2 x A1) - (O1 x A2)


(R1 x N2) - (R2 x N1)

x100


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.


Method for measuring cytotoxicity or proliferation using alamarBlue® by fluorescence

  1. Harvest cells which are in the log phase of growth and determine cell count. Adjust the cell count to 1x104cells/ml (suggested cell density). The optimum cell density may vary between cell types.
  2. Plate cells and expose to test agent as determined by researcher.  For determining the effect of a test agent on cell growth, ensure correct controls are included e.g. stimulated vs. unstimulated cells.
  3. Mix by shaking and then aseptically add alamarBlue® in an amount equal to 10% of the volume in the well. 
  4. Incubate cultures with alamarBlue for 4 - 8 hours. N.B. The optimum incubation time may vary between cell types. 
  5. Measure cytotoxicity or proliferation using spectrophotometry of fluorescence.
  6. Read fluorescence at excitation 560 nm, emission 590 nm. 

To calculate percent difference in reduction between treated and control cells in cytotoxicity/ proliferation assays use the following formula (Equation 4):  

Where: FI 590 = Fluorescent Intensity at 590 nm emission (560 nm excitation). 


Percentage difference between treated and control cells (equation 4)

=

FI 590 of test agent


FI 590 of untreated control

x100


 

If required, equation 2 can be used to find the percentage reduction of alamarBlue® using fluorescence: 

Where: FI 590 = Fluorescent Intensity at 590 nm emission (560 nm excitation). 


Percentage reduction of alamarBlue® (equation 2)

=

FI 590 of test agent - FI 590 of untreated control


FI 590 of 100% reduced alamarBlue® - FI 590 untreated control

x100


Example Data:

  • Media RPMI 1640
 
 
 
  • Fluorescence of untreated sample (positive control
=
25670
 
  • Fluorescence of treated sample
=
6472
 
Percentage difference between treated and untreated cells
=      
6472

25670
x 100

 

=
25%
 

For equation 2, it is necessary to include the fluorescence value for alamarBlue® in its fully reduced form. To produce the 100% reduced form of alamarBlue®, simply autoclave a sample containing media and alamarBlue for 15 minutes. Since fluorescence units are arbitrary and may therefore vary depending upon instrument set up, it is important that users determine the fluorescence reading for 100% reduction using the same media and instrument as for their samples.