Zinquin

Primary antibodies for β-actin (#12620), phospho (Thr202/Tyr204) ERK (#4370), and ERK (#9102), were from Cell Signaling Technology (Danvers, MA, USA). Antibodies for glutamic acid decarboxylase 65 (GAD65; SC-377154) and Tbr1 (SC-376258; Western blot) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for Neu-N (MAB377) and Tbr2 (AB15894) were from Millipore (Burlington, MA, USA). The antibody for Ki67 (550609) was obtained from BD Pharmingen (San José, CA, USA). Antibodies for Sox2 (ab97959), Tbr1 (ab31941; immunofluorescence), vGlut1 (ab77822) and Pax6 (ab5790) were from Abcam Inc. (Cambridge, MA, USA). Secondary fluorescent antibodies were obtained from Jackson ImmunoResearch Co. Laboratories (West Grove, PA, USA). Polyvinylidene difluoride (PVDF), membranes and molecular weight standards for Western blot were obtained from BIO-RAD (Hercules, CA, USA). The enhanced chemiluminescence (ECL) Western blotting system was from Thermo Fisher Scientific Inc. (Piscataway, NJ, USA). Zinquin, the antibody for γ amino butyric acid (GABA; A2052), and all other reagents were of the highest quality available and were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Zinquin (Sigma) was used to detect intracellular zinc levels after treatment with polaprezinc and ZnSO₄. Briefly, polaprezinc and ZnSO₄ were applied to hBMSCs or RAW264.7 cells. After 24 h, 25 μM of Zinquin was added and incubated for 30 min at 37 °C, and cells were washed three times with PBS to remove extracellular Zinquin. Fluorescence was measured at an excitation wavelength of 368 nm and emission of 490 nm using a Fluorometer (Varioskan Flash 3001, Thermo Fisher Scientific, Waltham, MA, USA).

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).