Limits...
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.


A microfluorometry system for multiparameric measurement. The diagram shows the instrumentation used to measure NADH (emission, 450 nm; excitation, 361 nm), Fura-2-FF (emission, 500 nm; excitation, 353 and 400 nm), and TMRE (emission, 590 nm; excitation, 530 nm) or carboxy-SNARF-1 (emission, 590 and 640nm; excitation, 539 nm). The light from a Xenon arc lamp excited the fluoroprobes in a myocyte, and then the emission passed through a dichroic mirror and band-pass filters to measure the intensity using photon-counting devices. Near infrared from a microscope was used as a light source to visualize the myocytes during experiments using a CCD camera and monitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499650&req=5

Figure 1: A microfluorometry system for multiparameric measurement. The diagram shows the instrumentation used to measure NADH (emission, 450 nm; excitation, 361 nm), Fura-2-FF (emission, 500 nm; excitation, 353 and 400 nm), and TMRE (emission, 590 nm; excitation, 530 nm) or carboxy-SNARF-1 (emission, 590 and 640nm; excitation, 539 nm). The light from a Xenon arc lamp excited the fluoroprobes in a myocyte, and then the emission passed through a dichroic mirror and band-pass filters to measure the intensity using photon-counting devices. Near infrared from a microscope was used as a light source to visualize the myocytes during experiments using a CCD camera and monitor.

Mentions: Ventricular myocytes were incubated in Fura-2-FF-AM (8 µM) solution for 60 min at 4℃, and then at 37℃ for 30 min. Next, ventricular myocytes were incubated for 60 min at 37℃ with a dye-free solution to sufficiently hydrolyze the AM ester dye. Finally, the ventricular myocytes were transferred into and incubated in fresh medium. If carboxy-SNARF-1 was needed for the study, the dye (2 µM) was added into the second incubation step. To remove cytosolic compartments and to visualize the mitochondrial fluorescence, single isolated cardiomyocytes were perfused with saponin (0.1 mg/mL) for 60 s in the perfusing bath which is mounted on the inverted microscope (Fig. 1). All experiments were performed at 37℃.


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)

A microfluorometry system for multiparameric measurement. The diagram shows the instrumentation used to measure NADH (emission, 450 nm; excitation, 361 nm), Fura-2-FF (emission, 500 nm; excitation, 353 and 400 nm), and TMRE (emission, 590 nm; excitation, 530 nm) or carboxy-SNARF-1 (emission, 590 and 640nm; excitation, 539 nm). The light from a Xenon arc lamp excited the fluoroprobes in a myocyte, and then the emission passed through a dichroic mirror and band-pass filters to measure the intensity using photon-counting devices. Near infrared from a microscope was used as a light source to visualize the myocytes during experiments using a CCD camera and monitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A microfluorometry system for multiparameric measurement. The diagram shows the instrumentation used to measure NADH (emission, 450 nm; excitation, 361 nm), Fura-2-FF (emission, 500 nm; excitation, 353 and 400 nm), and TMRE (emission, 590 nm; excitation, 530 nm) or carboxy-SNARF-1 (emission, 590 and 640nm; excitation, 539 nm). The light from a Xenon arc lamp excited the fluoroprobes in a myocyte, and then the emission passed through a dichroic mirror and band-pass filters to measure the intensity using photon-counting devices. Near infrared from a microscope was used as a light source to visualize the myocytes during experiments using a CCD camera and monitor.
Mentions: Ventricular myocytes were incubated in Fura-2-FF-AM (8 µM) solution for 60 min at 4℃, and then at 37℃ for 30 min. Next, ventricular myocytes were incubated for 60 min at 37℃ with a dye-free solution to sufficiently hydrolyze the AM ester dye. Finally, the ventricular myocytes were transferred into and incubated in fresh medium. If carboxy-SNARF-1 was needed for the study, the dye (2 µM) was added into the second incubation step. To remove cytosolic compartments and to visualize the mitochondrial fluorescence, single isolated cardiomyocytes were perfused with saponin (0.1 mg/mL) for 60 s in the perfusing bath which is mounted on the inverted microscope (Fig. 1). All experiments were performed at 37℃.

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.