Why is my background so high in my microplate cell-based assay?

June 25, 2012

Most likely your cell-culture media contains Phenol Red, which absorbs light in the visible range (400 to 600 nm).

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Dr EJ Dell
PhD, Sales Manager Northwest

To decrease the background, the Phenol Red must be washed off before measuring your cell-based assay. The best thing to do is to culture your cells in your microplate with Phenol Red. Then 1-2 hours before your assay measurement, wash off and replace the media + Phenol Red with media that has no Phenol Red. This will significantly decrease your background.


Another possibility is that the Gain is set too high. The Gain sets the electrical current across the photomultiplier tube (PMT) to change the sensitivity of the PMT. A lower Gain is used in assays that have higher concentrations of a signal, while a higher Gain is used in assays that have lower concentrations of a signal. Since cells have many different molecules that can absorb light, to keep the background low it is recommended to set the Gain on the blank well to 5% of the target value. For the Omega and PHERAstar series of readers, this would give a target value of around 13,000 RFUs for the blank (5% of 260,000 total possible RFUs); while for the OPTIMA and NOVOstar series of readers, this would give a target value around 3,250 RFUs for the blank (5% of 65,000 total possible RFUs). Then all other measurements should be higher than the blank at that Gain.

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