Fluo 3 am

Cells were loaded with 10 μM Fluo-3 AM (Life Technologies, CA, USA) using conventional protocols as recommended by the manufacturer. In brief, hMESCs were incubated with Fluo-3 AM for 60 min in Ca²⁺-free basic solution (144 mM NaCl, 4 mM KCl, 0.33 mM NaH₂PO₄, 5.5 mM glucose, 0.53 mM MgCl₂, 10 mM HEPES (Sigma-Aldrich, MO, USA), pH adjusted to 7.4 using NaOH) in the dark at room temperature. Then, Fluo-3 AM was washed out, and cells were incubated in the external basic solution with 4 mM CaCl₂ (Sigma-Aldrich, MO, USA) for another 15 min in the dark. Coverslips with Fluo-3-loaded cultures were placed in the perfusion chamber, which was mounted on the stage of Leica TCS SP5 MP inverted microscope (Leica Microsystems, Germany). Fluorescence was activated with 488 nm laser light and emission was measured within the wavelength range from 500 to 560 nm. Images were captured every 2.5 seconds during ∼ 60 min experiments.
In Ca²⁺ imaging experiments we used basic solution containing 144 mM NaCl, 4 mM KCl, 0.33 mM NaH₂PO₄, 5.5 mM glucose, 4 mM CaCl₂, 0.53 mM MgCl₂, 10 mM HEPES, pH adjusted to 7.4 using NaOH. In Ca²⁺-free experiments we used the same basic solution without CaCl₂. In order to avoid Ca²⁺ contamination in Ca²⁺-free experiments, basic solution was additionally supplemented with 4 mM EGTA (Sigma-Aldrich, MO, USA).

NNC 55-0396 hydrate (NNC-55) was purchased from Sigma-Aldrich (N0287). NNC-55 was dissolved in DMSO (Sigma-Aldrich, D2650) and stored at -20°C or -80°C. Other reagent sources are listed below: FBS, horse serum, trypsin/EDTA solution, DMEM (GIBCO-BRL, Gaithersburg, MD, USA); 3-Methyladenine (3-MA), hydroxychloroquine (HCQ), 4-phenylbutyric acid (4-PBA), Tauroursodeoxycholate (TUDCA) and collagenase (MCE, Monmouth Junction, USA); Annexin VPE/7-AAD Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA); Mitochondrial Permeability Transition Pore (mPTP) Assay Kit (YEASEN, Shanghai, China); Fluo-3AM (Sigma-Aldrich, 39294).

Cytosolic concentration of Ca²⁺ was monitored by measuring the fluorescence change of the Fluo-3 AM (Sigma-Aldrich, Cat. No. 73881) dye used as a probe for Ca^2+, whose intensity is directly representative of the cytosolic levels of the ion. Briefly, Fluo-3 AM at 2 µM final concentration was added to 1.0 × 10⁵ erythrocytes 40 min before the end of treatment in the dark. After centrifugation, (2000× g, 4 °C, 5 min) RBCs were washed, resuspended in PBS and analysed as reported in Section 4.4.3.

Dulbecco’s modified Eagle’s media (DMEM), Dulbecco’s phosphate buffer saline (DPBS), fetal bovine serum (FBS), penicillin–streptomycin, quercetin dihydrate (Qu), 3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT), propidium iodide, rhodamine 123, 2,7-dichlorodihydrofluoresceindiacetate (H₂DCF-DA), Fluo-3 AM, proteinase K, ribonuclease A, 1,2-bis-(O-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), β-nicotinamide adenine dinucleotide reduced disodium salt (β-NADH), ascorbic acid, agarose, dimethyl sulfoxide (DMSO) and anti-β-actin antibody were purchased from Sigma–Aldrich Chemicals (St. Louis, MO). Antibodies against Bcl-2, Bax, Bim, AIF, NF-κB, caspase-3/caspase-8, phospho-MEK, MEK1/2, phospho-ERK, ERK 1/2, phospho-p38, p-38, phospho-JNK, JNK, Nrf-2, catalase, Cu/Zn SOD, PI3K, phospho-Akt, Akt and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc,). FITC Annexin V was purchased from BD Pharmingen. Histone H3 antibody was purchased from Cell Signaling Technology.
Stock solution of quercetin (Qu) was prepared in dimethyl sulfoxide (DMSO) and diluted to the desired final concentration with culture medium just before use. The final DMSO concentration did not exceed 0.1% (v/v).

Changes in intracellular free Ca²⁺ were determined by single cell Fluo3 fluorescence imaging as previously described^{41 (link)}. The islets were incubated at 37 °C for 40 min with 2 mM Fluo3/acetoxymethyl ester (Fluo3-AM) (Sigma) supplemented with 0.0125% Pluronic F-127 (Sigma) in KRH solution containing 20 mM HEPES, pH 7.4, 2 mM CaCl₂, 3.3 mM glucose, followed by 10 min incubation at room temperature in KRH solution prior to imaging. The images were acquired every 10 s with a high resolution Retiga SRV CCD camera (QImaging) attached to a Zeiss Axiovert 200 inverted fluorescence microscope. The fluorescence signal of single cells was measured over time using the Image-ProPlus (Media Cybernetics) software program and the data were expressed as relative total fluorescence [F/F₀: ratio of fluorescence signal in each frame to basal value (F₀)] as a function of time^{41 (link)}.