Katrin von Daeniken coauthored the 2002 study showing interference of natural compounds with the MTT assay. Its cell-free tests showed that coloured formazan could form without living cells.[ref]
A cell-free negative control asks a different question from an untreated cell culture: can the test mixture generate the readout chemically? Both controls are useful because neither alone distinguishes every contribution to absorbance.
Consider an original four-condition example. After subtraction of the reagent background, cells alone give , compound alone gives , and cells plus compound give . If chemical and cellular contributions are additive, the corrected cellular signal is , or 70 percent of the untreated signal. The uncorrected ratio would be 110 percent. The subtraction is conditional: a compound may behave differently in a cell culture than in a cell-free mixture. An independent cell count therefore tests something that this arithmetic cannot guarantee.
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