Place the plate in a fluorescence plate reader and read the fluorescence intensity (RFU) at 540ex/590em. Return the plate to the incubator and incubate for 4 hours. 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. Depending on the toxicity of the compound, incubate at 37 ☌ 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).Īdd 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. 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). Seed the cells at 6000-8000 cells/100 µl/well in 96-well culture plates and let the cells grow overnight. 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).
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