= MTT formazan solubilization check
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An \b[MTT formazan solubilization check] tests whether the colored product formed from <MTT> has been recovered and dissolved consistently for the intended optical readout. It addresses a measurement stage between biochemical product formation and interpretation of <absorbance>, rather than directly testing <cell viability>.
The manufacturer describes formation of purple formazan crystals, followed by dissolution and multiwell optical measurement.<Cell Proliferation Kit I MTT instructions by Roche>[Section 3.1 and Figure 4, printed p. 9]{ref} A sample with undissolved particles is not necessarily equivalent to a homogeneous solution containing the same total product amount.
For an original model, let $n_F$ be total formed product and let $r$ be the <dissolved formazan recovery fraction>. With a suitable fixed-path solution measurement,
$$
A-A_0=\epsilon\ell\frac{rn_F}{V}.
$$
Two samples with equal $n_F$ but recovery fractions $0.6$ and $1$ give a signal ratio $0.6$ under these assumptions. Interpreting that ratio entirely as a difference in cell number would assign a handling effect to biology.
A controlled check can compare additional dissolution or mixing with a matched handling control while accounting for any change in volume, path length, temperature or solvent. In a chosen fixed-volume, fixed-path example, net absorbance rising from $0.24$ to $0.32$ after further handling means the initial signal was $75\%$ of the later one. It does not independently prove that the later measurement recovered every molecule.
A suitable known-product addition provides another test: the expected incremental signal is $\Delta A=\epsilon\ell\Delta n/V$ when the same solution model applies. A smaller observed increment can indicate loss, incomplete dissolution or a matrix-dependent optical response. The added product must be chemically appropriate and the solvent/geometry matched before calling the ratio a recovery estimate.
An apparent signal plateau is useful evidence of repeatability under that procedure, but can also occur because of an instrumental limit or another chemical change. The <formazan optical response> and <tetrazolium-reduction interference> should therefore be considered separately from product recovery. A reproducible dissolution step strengthens a readout model without making MTT color a direct cell count.
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