FAQ about AquaBluer
1. Should I keep AquaBluer™ in the freezer?
AquaBluer™ solution is stable at 4-22 °C for 12 months. However, if stored at –20 °C, its shelf life could extend indefinitely. It is important to keep the solution in complete darkness and minimize its light exposure.
2. How many cells should I seed in each well?
It depends on how fast the cells propagate and how long you need to expose the cells to a test compound. In most cases, it's a good bet to seed 6000-8000 cells/well. After overnight incubation, you will likely have a 20-30% confluent culture to start drug exposure and have ~72 hours of drug exposure period before the no-drug control start to enter apoptosis due to overgrowth.
3. Can AquaBluer™ treated culture be used for other assays?
AquaBluer™ is nontoxic to the cells and you should be able to use those cells for subsequent cell analysis. It is unlikely to interfere with other bioassays, but you need to test it for your particular assays to be sure. We routinely recover the media containing AquaBluer™ for multiplex cytokine ELISA assays.
4. I don’t have the exact 540ex/590em fluorescence filter set, what can I do?
You may use any fluorescence filter set covering 550±20nm for excitation and 600±20nm for emission. Similarly, for absorbance reading, you can use a 570±20nm and 600±20nm filter set.
5. My data does not fit the Four Parameter Model, what can I do?
If you do not include a wide enough range of test drug concentrations in your experiment or your test compound is too potent or too weak, your experiment may not generate data points around 90-100% or 1-10% viability range. In these cases, the Four Parameter Model, which assumes a sigmoid dose-response curve, may not be able to find the IC50. In such cases, you can either repeat the experiment to include enough higher or lower test drug concentrations, or if repeating the experiment is not practical you may use a theoretical -4 log concentration for 95% viability and/or a +4 log concentration for 5% viability to estimate the IC50 from your existing data points.