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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.


Cellular area-dependent autofluorescent background. ● (F361,450) was used for NADH signal correction. ○ (F353,500) and ▾ (F400,500) were used for Fura-2-FF signal correction, respectively. ▵ (F530,590) was used for TMRE signal correction.
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Figure 6: Cellular area-dependent autofluorescent background. ● (F361,450) was used for NADH signal correction. ○ (F353,500) and ▾ (F400,500) were used for Fura-2-FF signal correction, respectively. ▵ (F530,590) was used for TMRE signal correction.

Mentions: The excitation light intensity used to induce fluorescence was much stronger than the fluorescence emission. Although the selected excitation wavelength was far different from the emission wavelength and was blocked using a band-pass filter, the stray light from excitation was still present. The stray light was reflected at the boundary between the solution and the cover slip and diffracted in the cells due to cellular components. Cells may also produce unwanted autofluorescence. Those signal artifacts were comparable to the real fluorescence emission from the dye; thus, correction was necessary. First, the reflected signal at the cover slip boundary was measured. Then, the background signal due to cellular autofluorescence was measured in the presence of FCCP and in the absence of mitochondrial substrates. The cell-dependent background signal was linearly related to the cell area; these relationships are shown in Fig. 6. Later, the dye-loaded cellular area was measured, and the autofluorescence calculated from the cellular area was corrected. Further, the cell-free window background was also measured and subtracted. The background correction was performed before the experiments began. We determined the relationship between the cellular area and cellular autofluorescence on each experimental day.


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)

Cellular area-dependent autofluorescent background. ● (F361,450) was used for NADH signal correction. ○ (F353,500) and ▾ (F400,500) were used for Fura-2-FF signal correction, respectively. ▵ (F530,590) was used for TMRE signal correction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Cellular area-dependent autofluorescent background. ● (F361,450) was used for NADH signal correction. ○ (F353,500) and ▾ (F400,500) were used for Fura-2-FF signal correction, respectively. ▵ (F530,590) was used for TMRE signal correction.
Mentions: The excitation light intensity used to induce fluorescence was much stronger than the fluorescence emission. Although the selected excitation wavelength was far different from the emission wavelength and was blocked using a band-pass filter, the stray light from excitation was still present. The stray light was reflected at the boundary between the solution and the cover slip and diffracted in the cells due to cellular components. Cells may also produce unwanted autofluorescence. Those signal artifacts were comparable to the real fluorescence emission from the dye; thus, correction was necessary. First, the reflected signal at the cover slip boundary was measured. Then, the background signal due to cellular autofluorescence was measured in the presence of FCCP and in the absence of mitochondrial substrates. The cell-dependent background signal was linearly related to the cell area; these relationships are shown in Fig. 6. Later, the dye-loaded cellular area was measured, and the autofluorescence calculated from the cellular area was corrected. Further, the cell-free window background was also measured and subtracted. The background correction was performed before the experiments began. We determined the relationship between the cellular area and cellular autofluorescence on each experimental day.

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.