Fluo 3

It is well known that labeled Ca²⁺ indicators exhibit increases in fluorescence upon binding Ca²⁺. One of the indicators of that is Fluo-3, AM and it has been used to image the spatial dynamics of Ca²⁺ signaling. Changes in intracellular Ca²⁺ fluorescence concentration in Hep2 cell were monitored by fluorescence changes of Fluo-3 (Calbiochem, Darmstadt, Germany) as described in a previous study^{25 (link)}. First, the cells were seeded in 35 mm glass bottom dishes. Then, after incubating the cells with Fluo-3 (1 μM for one hour in the dark), the dye was excited by a 488 nm argon laser from the confocal microscope (LSM 800, Zeiss, Ankara, Turkey)^{25 (link)}. In some of the experiments, the cells were treated with TRPM2 antagonist (2-APB and 100 μM) to inhibit Ca²⁺ entry before CPx stimulation (1 mM)^{49 (link)}. The cells were analyzed at 515 nm by laser confocal microscopy fitted with a 40x oil objective. The results of fluorescence intensity in 10 μm² of cytosol were measured in the laser confocal microscope by using ZEN program. The mean fluorescence intensity was expressed with an arbitrary unit per cell.

The intracellular calcium of rat articular chondrocytes was monitored by fluorescence imaging [60 (link)]. Cells (2 × 10⁵) on coverslips were washed three times with D-Hanks’ soon and incubated with 0.02% Pluronic F-127 and 4 μM Fluo-3-AM (Biotium, Hayward, CA, USA) at 37 °C for 30 min. After incubation, the cells were washed twice with Hank’s solution to clear away extra Fluo-3-AM. To eliminate the effects of voltage-gated Ca²⁺ channels, ω-Conotoxin MVIIC (3 μM) and nimodipine (5 μM) were added to the extracellular solution. The fluorescence of intracellular Fluo-3 was measured by confocal laser scanning fluorescence microscopy (Carl-Zeiss, Jena, Germany) at a excitation wavelength of 488 nm and emission wavelength of 525 nm, respectively. Gray scale images were collected using fluorescence microscopy at different time points from 0 to 5 min and then archived as TIFF image files for later analysis. Significant images were analyzed with Leica-sp5 Leica Application Suite (LAS) AF software (Leica Microsystems Inc., Buffalo Grove, IL, USA).