4 bromo calcium ionophore a23187

Octanol, heptanol, carbenoxolone, oleamide, LaCl₃, Etd⁺, procion orange MX2R, brilliant blue G (BBG), Evans blue, propidium iodide, 4-Bromo calcium ionophore A23187 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The 9-amino-6-Chloro-2-Methoxyacridine (ACMA), DAPI⁺², hexidium iodide, nuclear yellow, BOBO 1-iodide, 7-aminoactinomycin D (7-ADD), YOYO 1-iodide, TOTO 1-iodide and ethidium homodimer–2–bromide obtained from Invitrogen (Massachusetts, USA), indocyanine green from Pulsion medical systems, (Feldkirchen, Germany) methylene blue from Euromed (Santiago, Chile).

SH-SY5Y cells plated in 96-well plates were washed once with mKRB media (in mM: 140 NaCl, 2.8 KCl, 2 MgCl₂, 10 HEPES, 2 CaCl₂, pH 7.4 at 37 °C) and incubated for 45 min, at 37 °C, with 2 mM Fura-2/AM (Invitrogen, Waltham, Massachusetts, USA catalog #F1221), 0.02% pluronic F-127 (Invitrogen, catalog # P3000MP) and 200 mM sulfinpyrazone (Sigma-Aldrich, catalog #S9509-5G) prepared in mKRB. After rinsing with fresh KRB, Fura-2 fluorescence was measured using a FLUOstar OPTIMA spectrofluorometer (BMG Labtech, Ortenberg, Germany) at dual 340/380 nm excitation and 510 nm emission wavelengths. Basal Fura-2 fluorescence was recorded every 20 s for 4 min followed by stimuli with FCCP/oligomycin (2/3.5 μM) (Sigma-Aldrich, catalog #C2920 and #75351) or thapsigargin (2.5 μM) (Sigma-Aldrich, catalog #T9033-.5MG), KCl (50 mM) and 4-Bromo-calcium Ionophore A23187 (2 μM) (Sigma-Aldrich, catalog # B7272). All plotted values were normalized for baseline values. In experiments with thapsigargin, Ca²⁺ was omitted from mKRB.

4-Bromo-calcium Ionophore A23187; DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate), DTNB (5,5-dithiobis (2-nitrobenzoic acid), or Ellman’s reagent), PBS (phosphate-buffered saline), HgCl₂, HT, Tyr and HA were from Sigma Chemical Co. (St. Louis, MO, USA), Annexin V-fluorescein isothiocyanate (V-FITC) Apoptosis Detection Kit (556547, BD Pharmigen, Franklin Lakes, NJ, USA) and Anti Glicophorin A antibody (FITC) were purchased from antibodies-online.com (ABIN6253946, antibodies-online GmbH, Aachen, Germany. https://www.antibodies-online.com/antibody/6253946/anti-Glycophorin+A+GYPA+antibody+FITC/ accessed on 12 April 2022). HT, Tyr and HA were prepared in dimethyl sulfoxide (DMSO) and diluted from 10 mM or 100 mM stock Solution. The resulting solutions were preventively tested upon RBC at their final concentrations to exclude any damage.