AquaBluer™ Cell Viability Assay Protocol
This protocol serves as an example to perform cytotoxicity assay using 1µl of AquaBluer™ for each well containing 100 µl of medium in a 96-well format (for 384-well and other formats, adjust the volumes accordingly).
1. Set up your 96-well culture plates
Seed the cells at 6000-8000 cells/100 µl/well in 96-well culture plates and let the cells grow overnight. Set up quadruplicate wells of 1) no-cell control (100 µl of medium, for background scattering subtraction), 2) vehicle control (100 µl of cells with the vehicle of test compound, as 100% viability), 3) positive control (optional, 100 µl of cells treated with a known cytotoxic compound), 4) test compound (100 µl of cells treated with 6-10 concentrations of 1:1 serially diluted test compound around its estimated IC50). Depending on the toxicity of the compound, incubate at 37 °C for 24-72 hours (inspect cell killing daily under microscope, when ~90% cell death is observed at the higher drug concentration(s), it will be a good time to perform the AquaBluer™ assay).
2. Perform the AquaBluer™ assay
Add 0.1 ml of AquaBluer™ to 10 ml of culture medium in a reagent reservoir, and pipette up and down 10 times to mix well. Aspirate to remove the medium from the cell culture and add 100 µl of the diluted AquaBluer™ to each well with a multi-channel pipettor. Return the plate to the incubator and incubate for 4 hours.
3. Raw data acquisition
Remove the plate from the incubator. Place the plate in a fluorescence plate reader and read the fluorescence intensity (RFU) at 540ex/590em.
4. Viability and IC50 calculation
Subtract the average of RFU of the No-cell control (background) from all other RFU values. Convert the test RFU values to % viability using the formula: % Viability = (RFUtest / RFUveh) x 100, where RFUveh is the average RFU of the No-drug wells. Enter the % viability values and corresponding log test compound concentrations into a non-linear regression program (GraphPad Prism) or free online IC50 calculator (ic50.tk) and use the Four Parameter Model to obtain the IC50 values and dose-response curve.
(Note: If a fluorescence plate reader is unavailable, you may use an absorbance plate reader to acquire AquaBluer™ viability data by recording A570 and A600 for each well at the end of the AquaBluer™ incubation period, subtracting each A600 value from its corresponding A570 value, and then use the difference (eq “RFU”) to calculate the % viability and then IC50 similarly.)