Cj. Goodwin et al., MICROCULTURE TETRAZOLIUM ASSAYS - A COMPARISON BETWEEN 2 NEW TETRAZOLIUM SALTS, XTT AND MTS, Journal of immunological methods, 179(1), 1995, pp. 95-103
Microculture tetrazolium assays are being widely exploited to investig
ate the mechanisms of both cell activation and cell damage. They are c
olorimetric assays which are based upon the bioreduction of a tetrazol
ium salt to an intensely coloured formazan. We contrast the responses
obtainable with two new tetrazolium salts, MTS and XTT, when used on t
he rat lymphoma cell line (Nb2 cells), which has been activated by hum
an growth hormone. These tetrazolium salts, unlike the more commonly u
sed MTT, form soluble formazans upon bioreduction by the activated cel
ls. This has the advantage that it eliminates the error-prone solubili
sation step which is required for the microculture tetrazolium assays
which employ MTT. Bioreduction of XTT and MTS usually requires additio
n of an intermediate electron acceptor, phenazine methosulphate (PMS).
We found that the XTT/PMS, but not the MTS/PMS, reagent mixture was u
nstable. Nucleation and crystal formation in the XTT/PMS reagent mixtu
re, prepared in DPBS, could occur within 1-3 min. This resulted in a d
ecline in XTT-formazan production and manifested itself in the microcu
lture tetrazolium assay as both poor within-assay precision and seriou
s assay drift. Several features of the system suggested that the forma
tion of charge-transfer complexes between XTT and PMS accounted for th
is instability. No such instability was encountered when MTS and PMS w
ere mixed. We demonstrate that MTS/PMS provides microculture tetrazoli
um assays for hGH which are free from these serious artefacts and whic
h are uniquely precise. In conclusion we therefore advocate the use of
MTS in preference to XTT for the new generation of microculture tetra
zolium assays.