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A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.

Lee JH, Ha JM, Leem CH - Korean J. Physiol. Pharmacol. (2015)

Bottom Line: With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM.However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+).Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Ulsan College of Medicine/Asan Medical Center, Seoul 138-736, Korea.

ABSTRACT
Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

No MeSH data available.


The excitation spectrum of NADH-Fura-2-FF. The spectrum shows the interference between NADH and Fura-2-FF at each emission (450 and 500 nm). (A) The NADH excitation spectrum at 450-nm emission. (B) The NADH excitation spectrum at 500-nm emission. (C) The Fura-2-FF excitation spectrum at 450-nm emission under Ca2+-free and 1 mM Ca2+ conditions. (D) The Fura-2-FF excitation spectrum at 500-nm emission under Ca2+-free and 1 mM Ca2+ conditions.
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Figure 2: The excitation spectrum of NADH-Fura-2-FF. The spectrum shows the interference between NADH and Fura-2-FF at each emission (450 and 500 nm). (A) The NADH excitation spectrum at 450-nm emission. (B) The NADH excitation spectrum at 500-nm emission. (C) The Fura-2-FF excitation spectrum at 450-nm emission under Ca2+-free and 1 mM Ca2+ conditions. (D) The Fura-2-FF excitation spectrum at 500-nm emission under Ca2+-free and 1 mM Ca2+ conditions.

Mentions: Simultaneous measurement of Fura-2-FF and NADH was technically difficult because each fluoroprobe produced signal interference. Fig. 2 shows this interference. Emission wavelengths of 450 and 500 nm were selected for NADH and Fura-2-FF, respectively. The excitation spectrums of both fluoroprobes overlapped. In addition, each fluoroprobe could generate emission at both 450 and 500 nm. The light intensity at each emission, therefore, contained signals from both fluoroprobes. This interference had to be corrected to obtain an exact measurement of the emission signal, specifically the quantitative measurement of Ca2+ with Fura-2-FF.


A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.

Lee JH, Ha JM, Leem CH - Korean J. Physiol. Pharmacol. (2015)

The excitation spectrum of NADH-Fura-2-FF. The spectrum shows the interference between NADH and Fura-2-FF at each emission (450 and 500 nm). (A) The NADH excitation spectrum at 450-nm emission. (B) The NADH excitation spectrum at 500-nm emission. (C) The Fura-2-FF excitation spectrum at 450-nm emission under Ca2+-free and 1 mM Ca2+ conditions. (D) The Fura-2-FF excitation spectrum at 500-nm emission under Ca2+-free and 1 mM Ca2+ conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499650&req=5

Figure 2: The excitation spectrum of NADH-Fura-2-FF. The spectrum shows the interference between NADH and Fura-2-FF at each emission (450 and 500 nm). (A) The NADH excitation spectrum at 450-nm emission. (B) The NADH excitation spectrum at 500-nm emission. (C) The Fura-2-FF excitation spectrum at 450-nm emission under Ca2+-free and 1 mM Ca2+ conditions. (D) The Fura-2-FF excitation spectrum at 500-nm emission under Ca2+-free and 1 mM Ca2+ conditions.
Mentions: Simultaneous measurement of Fura-2-FF and NADH was technically difficult because each fluoroprobe produced signal interference. Fig. 2 shows this interference. Emission wavelengths of 450 and 500 nm were selected for NADH and Fura-2-FF, respectively. The excitation spectrums of both fluoroprobes overlapped. In addition, each fluoroprobe could generate emission at both 450 and 500 nm. The light intensity at each emission, therefore, contained signals from both fluoroprobes. This interference had to be corrected to obtain an exact measurement of the emission signal, specifically the quantitative measurement of Ca2+ with Fura-2-FF.

Bottom Line: With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM.However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+).Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Ulsan College of Medicine/Asan Medical Center, Seoul 138-736, Korea.

ABSTRACT
Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

No MeSH data available.