MICROCULTURE TETRAZOLIUM ASSAYS - A COMPARISON BETWEEN 2 NEW TETRAZOLIUM SALTS, XTT AND MTS

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.