Calcium orange

The intracellular Ca²⁺ concentration in INS-1E cells was determined with a confocal laser scanning microscope and 5 μM calcium orange (MA, USA, Invitrogen). calcium orange was dissolved in Hanks buffer with additional Pluronic F-127 (0.005%, Molecular Probes, MA, USA) at 37°C for 30 min and subsequently washed twice using Hanks buffer. Fluorescence measurements were carried out using anargon ion laser (excitation: 561 nm). Images were recorded every 2 s using a DeltaVision Ultra system (GE, Massachusetts, USA). Baseline fluorescence (F0) was determined by averaging 20 images. Then cells were transferred in the KRBB buffer with 16.7mmol/l glucose, images were recorded every 2 s for 15min. Fractional fluorescence (F/F0) indicates the changes of intracellular calcium concentration. After the base line were stable for 5 min, the intracellular Ca²⁺ concentration was measured for another 5 min with Control (Ctrl), 10 mM tolbutamide (T), cisapride (C) or tolbutamide+cisapride (T+C) under the stimulation with different glucose stimulation. PBS was used as a control. AUC was measured before (0-5 min) and after (5-10 min) different drugs application. Statistical comparisons were calculated using the Wilcoxon matched pairs test (n=4).

The cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in primary hepatocytes was determined with a confocal laser scanning microscope and 5 μM calcium orange (MA, USA, Invitrogen). calcium orange was dissolved in Hanks buffer with additional Pluronic F-127 (0.005%, Molecular Probes, MA, USA) at 37°C for 30 min following washing with Hanks buffer twice.
To measure the mitochondrial Ca²⁺ concentration ([Ca²⁺]_m), the cells were seeded into a 15 mm cover glass-bottomed dish (NEST, Jiangsu, China) loaded with 4 μM Rhod-2/AM (Invitrogen) at 37°C and incubated for 30 min. Then, the cells were cultured for another 30 min at 37°C before analyzing on a confocal microscope (FV1000, Olympus, Japan) with an inverted microscope at 60x oil immersion objective.

Visualization of calcium ions in bacterial cells was performed by confocal microscope (Nikon Eclipse, Netherlands). Pigment solution was prepared in accordance with the recommendations of the manufacturer. Two millimolars of calcium orange (Invitrogen, USA) was dissolved in anhydrous DMSO. A small amount of bacteria (approx. 0.5 ml) was placed in an Eppendorf tube filled with 0.5 ml of PBS and then the microorganisms were dissolved in the solution by shaking. Twenty microliters of the prepared calcium orange solution was added to the bacterial suspension. The specimen for microscope observation was prepared by spreading the dyed bacterial suspension on a cover slide (without covering) and drying. An argon laser with a wavelength of 488 nm was used to induce fluorescence.