I accidentally froze the alamarBlue® stock reagent, can I still use it?
Yes. alamarBlue® reagent is stable to multiple freeze/thaw cycles. Be sure to heat the reagent in a 37°C water bath and mix the reagent to ensure a homogenous solution before use.
Do I need to protect alamarBlue® reagent from light?
Yes, alamarBlue® reagent is very slowly converted into a fluorescent product over time, when exposed to light, thus leading to high background values. Store the reagent, protected from light.
What is the optimal incubation time and temperature of cells with alamarBlue® reagent?
Incubate the cells with alamarBlue® reagent for 1–4 hours at 37°C. For more sensitive detection with low cell numbers, increase the incubation time for up to 24 hours.
Can you incubate cells with alamarBlue® reagent overnight?
Yes. However, signals from higher cell density samples may have “saturated,” which means the linearity of reagent may have reached a plateau. If this occurs, decrease the incubation time.
What if I don’t have an instrument suitable for reading fluorescence?
The absorbance of alamarBlue® reagent also changes depending on cell viability and proliferation. Therefore, simply monitor the absorbance of the reagent at 570 nm, while using 600 nm as a reference wavelength.
Is alamarBlue® assay strictly an endpoint assay?
No. While alamarBlue® can be used as a terminal readout of a population of cells, the reagent can also be used to continuously monitor cell viability and proliferation in real time. Since alamarBlue® reagent is non-toxic, you can incubate cells with reagent and monitor fluorescence (or absorbance) over time on the same sample.
What is the problem for observing high background fluorescence values?
The reagent may be breaking down due to exposure to light. Be sure to store alamarBlue® reagent in the dark and do not expose the reagent to direct light for long periods of time.
Why are the fluorescence values so low in intensity?
Try increasing the incubation time of cells with alamarBlue® reagent, changing the instrument’s “gain” setting, and checking the instrument filter/wavelength settings. Make sure to have positive controls (living cells) in the experimental design for troubleshooting.
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