Resazurin is reduced by cells to the fluorescent product resorufin. Resorufin can be reduced further to a non-fluorescent compound. Therefore the densest wells may have lowest fluorescence due to over-reduction of the substrate. Please see the product information sheet for more details. The kit protocol provides general guidelines and may need to be optimized empirically for your experimental system. You may need to vary cell density or assay incubation time to ensure that your samples fall in the linear range of the kit.
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