Confocal laser scanning fluorescence microscopy

Cells(2 × 10⁵)on coverslips were washed three times with Hank's solution and incubated with 4 μmol/L Fluo‐3‐AM and 0.02% Pluronic F‐127 (Biotium, Hayward, California, USA) for 30 minutes at 37°C. Cells were washed three times with Hank's solution at 25°C to remove the extracellular Fluo3‐AM. Nimodipine (5 μmol/L) was added to eliminate the effects of voltage‐gated Ca²⁺ channels from intracellular stores. Cells were perfused initially with D‐Hank's solution (pH6.5), PcTx1 and LY294002 with buffer containing PDGF (10 ng/ml). The fluorescence of intracellular Fluo‐3 was quantitated by confocal laser scanning fluorescence microscopy (Zeiss) with excitation at 488 nm and emission at 525 nm. Grey scale images with 0‐255 steps were collected at different time‐points before and up to 10 minutes after fluorescence microscopy and archived as TIFF image files for later analysis.

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).