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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 recommended that the plating density and incubation time be determined for each cell line using the following procedure:
(i) for any given incubation time selected, the range in cell density relating cell number to alamarBlue reduction can be determined by a linear response;
(ii) for any given cell density selected, the incubation time can be determined as the time taken for the control cells to turn the indicator from the oxidized (blue) form to the fully reduced (red) form.
a. Equation 1 calculates percentage reduction of alamarBlue using absorbance
Percentage reduction of alamarBlue (equation 1)
(O2 x A1) - (O1 x A2)
(R1 x N2) - (R2 x N1)
* Only one appropriate substitute wavelength may be used.
To calculate percent reduced using equation 1:
Molar extinction coefficients for alamarBlue at different wavelengths
b) To calculate percentage reduction of alamarBlue using fluorescence, use equation 2:
Percentage reduction of alamarBlue (equation 2)
FI 590 of test agent - FI 590 untreated control
FI 590 of 100% reduced alamarBlue - FI 590 untreated control
Where: FI 590 = Fluorescent Intensity at 590 nm emission (560 nm excitation).
a) For experiments to determine optimum cell density, plot log of cell density on the x axis, and percentage reduction on the y axis;
b) For experiments to determine the optimum incubation time, plot the number of hours on the x axis, and the percentage reduction on the y axis.
c) Use the graphs to determine optimum cell density or incubation period.