All Imaging Experiments Were Carried out at Room Temperature in a HEPES- And

Imaging experiments

All imaging experiments were carried out at room temperature in a HEPES-buffered salt solution (HCSS) containing, in mM: 140 NaCl, 5.4 KCl, 1 NaH2PO4, 1.8 CaCl2, 1 MgSO4,12 HEPES, and 5.5 D-glucose, pH 7.40.1. [h1]

Cells were loaded with fura-2 by 60 min incubation with 5 uM acetoxymethyl (AM) ester (Invitrogen, Eugene, OR) and 0.1% Pluronic F-127 (Invitrogen, Eugene, OR) in HCSS (pH=7.2) at room temperature, washed with HCSS and incubated for another 60 min to allow for the ester hydrolysis. After loading, the cells were imaged on an inverted microscope (Nikon Eclipse TE300, Nikon Inc., Melville, NY) using a 40x, 1.3 N.A fluorite oil immersion objective (Nikon) and a cooled CCD camera (Cooke Corp., Auburn Hill, MI) equipped with a 75 W xenon arc lamp. The fluorescence excitation was provided by a band-specific filters (340 and 380 nm; Semrock, Rochester, NY) in combination with XF73 dichroic beam splitter (Omega Optical, Brattleboro, VA). The pairs of images were collected at alternate excitation wavelengths at 10 sec intervals. After subtracting the matching background, the images were divided by one another to yield ratio values for individual cells.

Intracellular free Ca2+concentrations ([Ca2+]i) in single cells were calculated using the formula [1]

in which R was the measured fluorescence intensity ratio, RF and RB were the intensity ratios of free and Ca2+-bound indicator, and F2F/F2B was the correction factor defined as the second wavelength intensity of free and Ca2+-bound indicator. The calibration constants were determined in calibration solutions containing either 10 mM EGTA or 1 mM Ca2+in the same optical system. The MetaFluor software (Molecular Devices, Sunnyvale, CA) was used for image acquisition and analysis.

REFERENCES

1. G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J Biol Chem. 260 (1985) 3440-3450.

[h1]I do not recall the media the experiments were carried out - the description is of mine, Please change it accordingly if it was yours.