96 well flat clear bottom black polystyrene microplates

A fluorescent Zn assay was used to determine cytoplasmic levels of Zn. ARPE19 cells were cultured for 3 weeks in 96-well flat clear bottom black polystyrene microplates (Corning), Cells were washed twice with HBSS and incubated for 30 min at 37°C with HBSS containing FluoroZin3 (final 2.5 μM, Thermo Fisher), ER-Tracker Red (final 2 μM)and NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342) (both from Thermo Fisher) and washed twice with HBSS and replaced with FluoroZin3 (final at 2.5 μM, Thermo Fisher) in HBSS, with ER-Tracker Red (final at 2 μM) and NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342) (both from Thermo Fisher). Plates were washed with Hank’s Balanced Salt Solution (HBSS, Thermo Fisher) four times; RPMI 1640 Medium (no phenol red, Thermo Fisher) with ZnCl2 (5 μM) and sodium pyrithione (10 μM) (both from Sigma) was added. Plates were incubated for 2hr at 37°C. Live images were taken using IN Cell Analyzer 2200 (GE Healthcare Life Sciences). Co-staining of Zn and the endoplasmic reticulum were done by preloading ARPE-19 cells with endoplasmic reticulum (ER) specific sensor ZBR3 for 30min, wash thoroughly with HBSS, then add NVP-ZIP7-4 and zinc/pyrithione for 2 hr, before taking the live image using INCELL6500.

Free and encapsulated DyP peroxidase (KpDyP and KpDyP_Enc) were purified and diluted to equimolar heme concentrations as determined via heme assay kit (Sigma-Aldrich, USA) per manufacturer protocol to analyze enzyme activity via ABTS assays. Reactions contained 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer and 150 mM NaCl at pH 5.5, with 5 mM ABTS and each respective enzyme (free or encapsulated) standardized to 600 nM heme concentration. Reactions were initiated by the addition of varying concentrations of hydrogen peroxide (H₂O₂) (two-fold dilutions for final reaction concentrations ranging from 10 mM to 1.2 μM). The oxidation of ABTS was measured at 420 nm in a BioTek Synergy H1 microplate reader at a final volume of 100 μL in Corning 96-well flat clear bottom black polystyrene microplates in 10 s intervals for a total of 15 min to evaluate enzyme activity and the rate of reaction. All assays were conducted in triplicate. Non-linear regression curve analysis with Michaelis-Menten fit of initial velocities was conducted with GraphPad Prism 10.