I incubated HRECs with RIG012 at a 1% dilution (1 μL of RIG012 per mL of culture medium) for 24 hours; this concentration, hereafter referred to as 1x, has been previously shown to exert inhibitory effects in Western blotting. For the cytotoxicity assessment using the CCK8 assay, I established four concentration gradients (4x, 2x, 1x, and 0.5x) based on the 1x working concentration used in Western blotting.
Notably, the absorbance results obtained one hour after the assay showed no significant differences between the treated groups and the control group. To minimize the impact of extreme values, each concentration was tested in ten replicates. It should be noted that due to the lack of relevant literature on CCK8 experiments with RIG012, these concentration gradients were set arbitrarily. Additionally, by the second hour, nearly all CCK8 absorbance values remained below 2, suggesting that RIG012 did not exhibit cytotoxicity toward HRECs.
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Not all "effective" CCK8 drugs can detect a decrease in O.D., especially for drugs that inhibit mitochondrial respiration or cellular metabolism without directly impairing them. It's recommended to use multiple time points (24, 48, or even 72 hours) and conduct cross-validation with multiple methods. Furthermore, Western blotting (WB) reflects molecular events, while CCK8 more closely reflects physiological energy status, and there can sometimes be a disconnect between the two.
I haven't found any literature that matches your experimental design; except for animal studies, the relevant literature uses direct cell-based WB or PCR:
PMID: 38316991 and PMID: 39214979
Note: RIG012 primarily targets RIG-I and is more effective in cells with high RIG-I expression.
The answer to this question comes from MCE Technical Support.
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