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 |
= |
(O2 x A1) - (O1 x A2) (O2 x P1) - (O1 x P2) |
x100 |
---|
Where:
* Only one appropriate substitute wavelength may be used.
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) |
---|---|---|
540 nm | 104395 | 47619 |
570 nm | 155677 | 80586 |
600 nm | 14652 | 117216 |
630 nm | 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:
* Only one appropriate substitute wavelength may be used.
To calculate percentage 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 |
= |
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 |
---|
|
|
|
|
|
=
|
25670
|
|
|
=
|
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 min. 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.