Using the data from Figure 1b: If, for example, the desired incubation time with alamarBlue® were 4 hours, any plating density from 500 to 10,000 cells/ml could be used and expected to produce a reaction within the linear range for alamarBlue® for that incubation period with a549 cells. However, if the intent is to incubate for 20 hours, the reaction could only be expected to be within the linear range if plated at 500-1000 cells/ml for this cell line.
On the other hand, for p388, even with an initial plating density up to 10,000 cells per well, data is within range and alamarBlue® has only been reduced by 50% after 20 hours. This indicates cells should be incubated with alamarBlue® for up to 2 days, even when plated at this high density. If a shortened exposure is desired, then the initial plating density should be increased. If the goal is to continue the experiment for more than 2 days, then the initial plating density should be decreased. Similar information is gained from examination of Figures 2a & b.
Note: With high cell numbers or extended incubation time (days), a point will be reached where the red form stops increasing and begins to decline. The absorbance/fluorescence level drops with a corresponding clearing of the red color. This is demonstrated in Figure 1a, when a549 cells are plated at 10,000 cells/ml and incubated for more than 4 hours. Refer to Frequently Asked Question 7 for further information about extended incubation times.