Fluo 4 acetoxymethyl ester fluo 4 am

Hesperidin (Figure 1a), glucose, N−acetylcysteine (NAC), 2′,7′−dichlorodihydrofluorescein diacetate (H₂DCFDA), avidin−tetramethyl−rhodamine isothiocyanate (TRITC) conjugate, and actin were obtained from Sigma−Aldrich (St. Louis, MO, USA). 5,5−Dimethyl−1−pyrroline−N−oxide (DMPO) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Fluo−4 acetoxymethyl ester (Fluo−4 AM) and Rhod−2 AM were purchased from Molecular Probes (Eugene, OR, USA). Hoechst 33342 was provided by Biomol GmbH (Hamburg, Germany). Inhibitors of U0126 and SP600125 were provided from Merck KGaA (Darmstadt, Germany) and Tocris (Ellisville, MO, USA), respectively. Primary antibodies against phospho−H2A.X (Ser139), C/EBP homologous protein (CHOP), caspase−9, caspase−3, phospho−extracellular signal−regulated kinase (ERK), phospho−c−Jun N−terminal kinase (JNK), and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against Bcl−2, Bcl−2−associated X protein (Bax), phospho−protein kinase R−like ER kinase (PERK), phospho−eukaryotic initiation factor 2 alpha (eIF2α), and ERK2 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti−IgG secondary antibodies were purchased from Invitrogen (Rockford, IL, USA). All other chemicals and reagents used were of analytical grade.

The hIOs were treated for 1 h with 5 μM Fluo-4 acetoxymethylester (Fluo-4 AM; Molecular Probes, Eugene, Oregon, USA) and washed with Ca²⁺-free isotonic buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl₂, 5.5 mM D-glucose). The hIOs were mounted on a confocal microscope (FV1000 Live; Olympus) and stimulated with 50 mM glucose (Sigma-Aldrich) in Ca²⁺-free isotonic buffer. hIOs were excited at 488 nm, and the signal emitted at 505 to 530 nm was recorded. The fluorescence intensity of the region of interest (ROI) was calculated using FV1000 software.

Flow cytometry was used to confirm the intracellular Ca²⁺ concentration in Min6 cells. Min6 cells were divided into groups and cultured alone, cultured with ISC cell supernatant, or cultured with exogenous Wnt5a (0.05 μg/ml) for 48 h. Then, the cells were trypsinized, washed, and incubated in complete DMEM containing 5 μM Fluo-4-acetoxymethyl ester (Fluo-4 AM; Molecular Probes, BD, USA) and 3% dimethyl sulfoxide at 37°C for 20 min. After incubation, the cells were washed three times with PBS by centrifugation. The cells were then resuspended in PBS. For each sample, fluorescence was analysed using a 530/30 filter.

Chemicals. We obtained lead(II) acetate (Pb²⁺), calcium chloride, glutaraldehyde solution, osmium tetroxide, bovine serum albumin (BSA), HEPES, sodium citrate, dimethyl sulfoxide (DMSO), isopropanol, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and dihyroethidium (DHE) from Sigma Chemical Co. (St. Louis, MO, USA). Phycoerythrin (PE)-labeled monoclonal antibody against human glycophorin A (anti-glycophorin A-RPE) was from Dako (Glostrub, Denmark), and fluo-4 acetoxymethyl ester (Fluo-4 AM) was from Molecular Probes (Eugene, OR, USA). We obtained fluorescein isothiocyanate (FITC)-labeled annexin V (annexin V-FITC) from Pharmingen (San Diego, CA, USA) and 5-(-6)-carboxyfluorescein diacetate (CFDA), calcein red-orange, anti-CD13-PE, anti-CD13-perCP-Cy5.5, and H2-DCFDA from Invitrogen (Eugene, OR, USA). Keratinocyte serum-free media kit was from Gibco BRL Life Technologies Inc. (Grand Island, NY, USA). All other reagents were of the highest purity available.

hIOs were loaded with Fluo-4 acetoxymethylester (fluo-4AM, 5 μM for 1 h, Molecular Probes, Eugene, Oregon, USA). hIOs were then washed with Ca²⁺-free isotonic buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 5.5 mM D-Glucose, 2 mM MgCl₂) and placed on the stage of confocal microscope (FV1000 Live, Olympus). hIOs were stimulated with 50 mM glucose (Sigma Aldrich) in Ca²⁺-free isotonic buffer. hIOs were excited at 488 nm, and the signal emitted at 505–530 nm was recorded. The fluorescence intensity of the region of interest (ROI) was calculated using FV1000 software.