Alamar Blue Assay Lab Report

After Alamar Blue dye is added, the plate is incubated for 4 hours and then is observed under a spectrophotometer. The spectrophotometer can measure fluorescence of the dye at 570 nm, which is the wavelength of the fluorescence of Alamar Blue dye. The spectrophotometer will return values for each well that correlates with the measured absorbance. These absorbance values can be converted to survivability percentage and can be graphed with a GraphPad program as shown in Graph 1A and 1B. The software can calculate survivability of cells by normalizing the absorbance value of the experimental group with absorbance value of the positive control group (Larson et al., 1997).Alamar Blue Assay detects cell viability through the compound resazurin in the Alamar Blue dye. Resazurin in its natural state is a blue non-fluorescing compound, but when reduced, resazurin fluoresces. This

The dye is simple to use, as it detects cell viability by just the application of the dye onto the cells. Furthermore, the dye is non-toxic, meaning the assay is not an end point assay. Therefore, the cells plated onto the plate would still be viable for use in further experiments. The assay is also very sensitive, and can detect down to 50 mammalian cells. The downside to this technique is that the fluorescence is dependent on temperature and pH of the solution, so in order to obtain an accurate reading of the plate, the plate would have to be at a consistent condition. To prevent this issue, the plate would be incubated throughout the majority of the experiment. (Obrien et al., 2000) Another weakness is the possibility of false positives or negatives that would result from the drug inhibiting or inducing enzymatic activity that fluoresces the Alamar Blue dye without actually altering viability of the cells (Quent et al.,