Multiplexed experiments

HEK293 cells were co-transfected with pC1-oROS-HT/pC1-SypHer3s or pC1-oROS-HT-C199S/pC1-SypHer3s as described above. 2 days post-transfection, both sensors expressed in HEK293s were imaged using epifluorescence microscope. pH change experiment for oROS-HT-C199S were performed with HEK293s in PBS (10010001, Gibco) prepared at pH of 6, 7.44, and 9. Fluorescence level for GFP and Far-red profile were captured every 1.5 seconds. Sequential pH-changes plus Menadione applications were performed with HEK293s in PBS (pH 7.44), which was changed to PBS (pH 6) followed by menadione stimulation prepared in PBS (pH 6). Fluorescence level for GFP and Far-red profile were captured every 2 seconds.

HEK293 cells were co-transfected with pC1-oROS-HT and pC1-Grx1-roGFP2 as described above. 2 days post-transfection, both sensors expressed in the cells with live cell imaging solution with 10mM glucose (LCIS+, Gibco, A14291DJ) were imaged using an epifluorescence microscope. For the sequential response of oROS-HT/Grx1-roGFP2 to 10µM H₂O₂, fluorescence level for GFP and Far-red profile were captured every second. For the response to Auranofin, fluorescence levels for GFPratio, GFP, and Far-red profiles were captured every minute.

hiPSC-CMs were transfected with pCAG-oROS-HT as described above. 2 days post-transfection, cells were incubated with Fluo-4 (Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"F14201","term_id":"860754","term_text":"F14201"}}F14201) at 5µM and JF635-HTL in RPMI + B27+insulin for 1 hour prior to imaging. For the response to Auranofin, fluorescence level of GFP profile (10Hz) and Far-red (0.1Hz) profile were acquired every 10 seconds for hiPSC-CMs in HEPES-buffered RPMI + B27+insulin.