Fluozin 3am

Cells were seeded and grown in multi-well 24 plates until reaching 80% of confluence. Cells were incubated with 1 µM of FluoZin-3AM (Invitrogen, Darmstadt, Germany) or 25 µM of Zinquin (Sigma-Aldrich, Darmstadt, Germany) for 30 min at 37 °C (5% CO₂) in isotonic solution containing (in mM) 140 NaCl, 5 KCl, 1.2 CaCl₂, 0.5 MgCl₂, 5 glucose, and 10 Hepes (300 milliosmoles/liter, pH 7.4), plus different concentrations of Zn²⁺ and/or chloroquine (CQ).Cells were then dissociated with Trypsin 0.05% in 0.53 mM EDTA, and were washed with PBS 1x. Fluorescence was quantified using an LSRII flow cytometer. Further analysis was performed using Flowing software (Perttu Terho, Turun yliopisto, Turku, Finland).
For in vivo confocal imaging, cells grown on 22 mm coverslips were incubated with Lysotracker, together with FluoZin-3AM or Zinquin, in a solution with 50 µM Zn²⁺ for 30 min; they were then washed twice with PBS and placed under the microscope in isotonic solution for imaging with an SP8 Leica microscope (Wetzlar, Germany).

To monitor the basal (resting) level of intracellular‐free Zn²⁺ ([Zn²⁺]_i) in H9c2 cells, we used a Zn²⁺ sensitive fluorescence dye‐loaded cells, using non‐ratiometric FluoZin‐3 (3‐μM FluoZin‐3 AM) for confocal microscope (LEICA SP5). Florescence intensities were acquired at 1 Hz, 490 nm excitation wavelength and collected at 525 nm. Image analysis was performed with ImageJ software. The steady state fluorescence intensity (F) was measured, then maximum and minimum ratios were determined to calculate free Zn²⁺ level using the following formula: [Zn²⁺] = K_d(F−F_min)/(F_max−F), where the K_d for FluoZin‐3 is 15 nM. The maximum fluorescence (F_max) was obtained upon Zn²⁺ saturation with Zn²⁺ salt of 1‐hydroxypyridine‐2‐thione, Zn²⁺‐pyrithione (Zn²⁺/Pyr; 10 μM), and the minimum ratio (R_min) was obtained upon intracellular Zn²⁺ chelation with N, N, N', N'‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN; 50 μM).

HT29 cells (ZnT2-attenuated or control) were cultured on glass coverslips in 24 well plates for 48 h. Cells were washed with 1X PBS and treated with FluoZin3-AM (ThermoFisher, Waltham, MA, USA, F24195; emission 485 nm/excitation 520 nm) in Opti-MEM containing 0.02% Pluronic F-127 (ThermoFisher, Waltham, MA, USA, P3000MP) for 1 h at 37 °C, as recommended by the manufacturer. Subsequently, cells were rinsed twice with PBS for 5 min each at RT with constant shaking. The fluorescence of FluoZin3-AM in live cells was imaged using the Leica Inverted Confocal Microscope SP8 (magnification = 63× oil) in three random images from three different samples/group. The experiment was repeated three times. As a negative control, HT29 cells were treated with the Zn chelator, N, N, N’, N’ -tetrakis (2-pyridylmethyl)ethylenediamine (TPEN; 10 µM, ThermoFisher, Waltham, MA, USA) for 30 min prior to FluoZin3-AM treatment.