Cell-free reductant profiling. To report on a protein selectively in cellular settings, a trigger must be completely resistant to reduction by the mM cellular monothiol background. Resistance or sensitivity to a reductant is best addressed by dose-response functions over time. Therefore, to best characterise the response to reductant challenges, we titrated reductants up to supraphysiological concentrations, with full timecourse rather than endpoint data. We began with GSH challenge tests. Over 6 h, A-type probes showed zero fluorescence response to 10 mM GSH, indicating outstanding monothiol resistance (Fig. , Fig. ). The thermodynamically identical G-type controls were GSH-labile (3 mM GSH activated to ca. 25%, Fig. ), supporting our pathway-based design (Fig. ). Dose-response plots of GSH resistance with the 6 h timepoint data (Fig. , Fig. ) highlight the GSHrobustness of all A-type probes and the partial GSH-lability of Gtype pathway controls. The comparison probe types behaved as expected. We had hoped that cyclic disulfides S1-S2, analogously to previously reported vicinal-dithiol-selective probe S3, would be monothiol resistant (i.e. no destabilisation by the solubiliser sidechains); they indeed resisted GSH up to 10 mM (Fig. ). In comparison to these, linear disulfide SS00 is a mechanistic control for the need of a cyclic dichalcogenide topology (Fig. ); its sensitivity to monothiol-based release was striking (nearly full activation by 1 mM GSH; Fig. , Fig. ). The cyclic diselenide Se3 was nonreactive (Fig. ), suggesting that selenium is tolerated at both positions of the reduction-based probes without Se-oxidation-mediated release mechanisms (see Supporting Note 2). The zero-level fluorescence seen with RX1 (and Se3) under a thousandfold challenge by GSH also qualifies the secondary amine phenolic carbamate design as exceptionally stable to monothiolysis, hydrolysis and aminolysis.