期刊
KOREAN JOURNAL OF PHYSIOLOGY & PHARMACOLOGY
卷 19, 期 4, 页码 373-382出版社
KOREAN JOURNAL OF PHYSIOLOGY & PHARMACOLOGY
DOI: 10.4196/kjpp.2015.19.4.373
关键词
Calcium; Fura-2-FF; Mitochondrial membrane potential; NADH; pH
资金
- National Research Foundation of Korea (NRF) - Ministry of Education, Science and Technology [2011-0010965, 2012-0009829]
- National Research Foundation of Korea [2011-0010965, 2012-0009829] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca2+]. 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 [Ca2+], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca2+] 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, [Ca2+], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca2+] and TMRE for Psi(m) or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca2+] concentration was 1.03 mu M. This 1 mu M cytosolic Ca2+ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca2+] increase was limited to similar to 30 mu M in the presence of 1 mu M cytosolic Ca2+. 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.
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