N 2.1 filter cube

100 μM of resorufin (Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) solution was prepared in Milli-Q water for the generation of a standard curve. Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit was obtained from Invitrogen (Invitrogen, Fisher Scientific, Landsmeer, The Netherlands). Stock solutions of 100 U/mL of HRP and 2 mM of AR were prepared in reaction buffer (0.25 M sodium phosphate, pH 7.4) and stored at -20°C. The stock solutions were diluted with reaction buffer immediately before use. 30% of hydrogen peroxide solution was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands) and diluted with Milli-Q water before use. The fluorescent signal from resorufin was observed by a Leica N 2.1 filter cube (excitation: BP 515–560 nm; emission: LP 590 nm). For the off-chip measurement of the resorufin fluorescent signal we used a spectrometer (Maya 200 Pro, Ocean Optics BV, Duiven, The Netherlands) with a xenon light source (HPX-2000, Ocean Optics BV, Duiven, The Netherlands) and a cuvette (fluorescence cell, 105.251-QS, Hellma BV, Nieuwegein, The Netherlands).

Preliminarily, a range of four ReAsH-EDT2 doses (1.25 μM, 2.5 μM, 5 μM, and 10 μM) was tested on L. lactis NpB to investigate saturation uptake. A preliminary test for the necessity of BAL treatment was also performed on NpB cells. Roughly 4 × 10⁹ cells of each sample were washed twice with PBS before being treated with 500 mL 2,3-Dimercaptopropanolin (BAL) 650 μM in PBS for 30 min. The cell pellet was then washed twice with PBS and stained with 2.5 μM ReAsH-ETD2 (Cayman, Ann Arbor, MI) in PBS for 30 min. After staining, the cell pellet was washed two consecutive times with 500 μL BAL 250 μM in PBS before finally being dissolved in 100 μL PBS. All the incubations were maintained at 37 °C. Unstained cells, stained empty-vector cells, and stained wildtype were processed in the same procedure as negative controls. The samples were screened under a live-cell epi-fluorescence microscopy system (Leica DMI6000B), lamp EL6000, objective HCX PL APO 63×/1.40 oil with Leica N2.1 filter cube (BP 515–560). The cells can be screened in real-time or stored at 4 °C until screening time.