This work was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1A6A3A01011832), by the Ministry of Science, ICT &#38; Future Planning (NRF-2016M3C1A6936606) and by the Ministry of Trade, Industry &#38; Energy (10068076).

All experimental protocols were approved by the local institutional animal care and use committee.
1. Solution preparation
<ol>
	<li>Prepare single freshly isolated cardiac myocytes11. 
	NOTE: Each laboratory might have a different cell storage solution. Here, the myocytes are stored in culture medium (DMEM).</li>
	<li>Prepare 100 mL of Ca2+-free solution (Table 1).</li>
	<li>Prepare 50 mL of culture medium in a 50 mL beaker. Aliquot 5...

<ol>
	<li>Berridge, M. J., Bootman, M. D., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling:+dynamics,+homeostasis+and+remodelling.">Calcium signalling: dynamics, homeostasis and remodelling.</a> Nature Reviews Molecular Cell Biology. 4 (7), 517-529 (2003).</li><li>Miyata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+free+Ca2++concentration+in+living+single+rat+cardiac+myocytes.">Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes.</a> American Journal of Physiology. 261 (4 Pt 2), H1123-H1134 (1991).</li><li>Allen, S. P., Stone, D., McCormack, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+loading+of+fura-2+into+mitochondria+in+the+intact+perfused+rat+heart+and+its+use+to+estimate+matrix+Ca2++under+various+conditions.">The loading of fura-2 into mitochondria in the intact perfused rat heart and its use to estimate matrix Ca2+ under various conditions.</a> Journal of Molecular Cellular Cardiology. 24 (7), 765-773 (1992).</li><li>Griffiths, E. J., Halestrap, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrophosphate+metabolism+in+the+perfused+heart+and+isolated+heart+mitochondria+and+its+role+in+regulation+of+mitochondrial+function+by+calcium.">Pyrophosphate metabolism in the perfused heart and isolated heart mitochondria and its role in regulation of mitochondrial function by calcium.</a> Biochemical Journal. 290 (Pt 2), 489-495 (1993).</li><li>Crompton, M., Moser, R., Ludi, H., Carafoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interrelations+between+the+transport+of+sodium+and+calcium+in+mitochondria+of+various+mammalian+tissues.">The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues.</a> European Journal of Biochemistry. 82 (1), 25-31 (1978).</li><li>Chance, B., Schoener, B., Oshino, R., Itshak, F., Nakase, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation-reduction+ratio+studies+of+mitochondria+in+freeze-trapped+samples.+NADH+and+flavoprotein+fluorescence+signals.">Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.</a> The Journal of Biological Chemistry. 254 (11), 4764-4771 (1979).</li><li>Eng, J., Lynch, R. M., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinamide+adenine+dinucleotide+fluorescence+spectroscopy+and+imaging+of+isolated+cardiac+myocytes.">Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.</a> Biophysical Journal. 55 (4), 621-630 (1989).</li><li>Brandes, R., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurements+of+mitochondrial+NADH+and+Ca(2+)+during+increased+work+in+intact+rat+heart+trabeculae.">Simultaneous measurements of mitochondrial NADH and Ca(2+) during increased work in intact rat heart trabeculae.</a> Biophysical Journal. 83 (2), 587-604 (2002).</li><li>Jo, H., Noma, A., Matsuoka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-mediated+coupling+between+mitochondrial+substrate+dehydrogenation+and+cardiac+workload+in+single+guinea-pig+ventricular+myocytes.">Calcium-mediated coupling between mitochondrial substrate dehydrogenation and cardiac workload in single guinea-pig ventricular myocytes.</a> Journal of Molecular Cellular Cardiology. 40 (3), 394-404 (2006).</li><li>Lee, J. H., Ha, J. M., Leem, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Nicotinamide+Adenine+Dinucleotide+Correction+Method+for+Mitochondrial+Ca2++Measurement+with+FURA-2-FF+in+Single+Permeabilized+Ventricular+Myocytes+of+Rat.">A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.</a> Korean Journal of Physiology and Pharmacology. 19 (4), 373-382 (2015).</li><li>Powell, T., Terrar, D. A., Twist, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+properties+of+individual+cells+isolated+from+adult+rat+ventricular+myocardium.">Electrical properties of individual cells isolated from adult rat ventricular myocardium.</a> Journal of Physiology. 302, 131-153 (1980).</li><li>Sun, B., Leem, C. H., Vaughan-Jones, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+chloride-dependent+acid+loader+in+the+guinea-pig+ventricular+myocyte:+part+of+a+dual+acid-loading+mechanism.">Novel chloride-dependent acid loader in the guinea-pig ventricular myocyte: part of a dual acid-loading mechanism.</a> Journal of Physiology. 495 (Pt 1), 65-82 (1996).</li><li>Page, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+ultrastructural+analysis+in+cardiac+membrane+physiology.">Quantitative ultrastructural analysis in cardiac membrane physiology.</a> American Journal of Physiology. 235 (5), C147-C158 (1978).</li><li>Page, E., McCallister, L. P., Power, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterological+measurements+of+cardiac+ultrastructures+implicated+in+excitation-contraction+coupling.">Sterological measurements of cardiac ultrastructures implicated in excitation-contraction coupling.</a> Proceedings of the National Academy of Sciences of the United States of America. 68 (7), 1465-1466 (1971).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> The Journal of Biological Chemistry. 260 (6), 3440-3450 (1985).</li></ol>

The interference correction method was successfully developed for measuring the signals of NADH and fura-2 analogs. Exact measurement of the signals is essential for exact correction. However, the inherent nature of the fluorescent device produces a background signal unrelated to that of NADH of fura-2. The highest quality band-pass filter can only pass up to 10&#8722;8 of the unwanted wavelengths of the light. However, the fluorescent signal from a single cell is very small, and the reflection of the excitati...

The significant role of intracellular Ca2+ is widely known1. The quantification of [Ca2+] is essential to understand the processes of the cellular physiological functions. Fura-2 analogs are quite useful because they are excited in the UV range (&#60;400 nm), and the ratiometric method can be applied for the quantitative measurement. Therefore, other physiological parameters such as pH, membrane potential, etc., can be measured with other fluorescent dyes. The mitochondrial Ca2+ concentration ([Ca2+]m) range was reportedly 0.08&#8722;20 &#956;M2,3,4,5. Among fura-2 analogs, fura-2-FF is appropriate for measuring this range of [Ca2+]. However, the live cells unfortunately contain NADH/NADPH for their metabolic processes, and NADH generates signal interference because of the overlapping excitation and emission spectra with the fura-2 analog. This interference greatly limits the use of fura-2 analogs. Specifically, if the analog is applied to measure mitochondrial [Ca2+], this interference is the biggest obstacle because the highest amount of NADH is in the mitochondria. This is further complicated by NADH changes being related to the mitochondrial membrane potential (&#936;m) and the change of &#936;m affects [Ca2+]m6,7,8,9. Furthermore, for studying [Ca2+]m dynamics, it is essential to know the status of other mitochondrial parameters, such as NADH, &#936;m, and pH.
The emissions at 450 nm and 500 nm with excitations at 353 nm, 361 nm, and 400 nm contain the signals from NADH and fura-2-FF, and the equations are as follows. Herein, 353 nm and 361 nm are the isosbestic points of fura-2-FF for emissions at 450 nm and at 500 nm, respectively.
F361,450 = F361,450,NADH + F361,450,Fura&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 1 
F353,500 = F353,500,NADH + F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 2 
F400,500 = F400,500,NADH + F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 3
where Fx,y is the measured emission intensity at y-nm by x-nm excitation, Fx,y,NADH represents the pure NADH-dependent emission intensity, and Fx,y,Fura represents the pure fura-2-FF-dependent emission intensity. Under the same concentration of the fluorescent dye, a certain excitation intensity should produce the same emission intensity. Therefore, the emission intensity ratio of two different excitation wavelengths should be constant. Ca2+ and fura-2 did not affect NADH fluorescence characteristics; therefore, the ratio of the emission at 450 nm and at 500 nm of NADH was constant at any excitation wavelength. The same rule can be used for fura-2-FF based on the assumption that NADH or [Ca2+] does not affect the emission and excitation spectra of fura-2-FF. However, Ca2+ caused a spectral shift of the fura-2-FF emission. Therefore, to remove the effect of Ca2+, isosbestic excitation, which is independent of Ca2+, needs to be used. Each emission wavelength (i.e., 450 nm and 500 nm) has a different isosbestic point, and from our experimental setup, 353 nm at 500 nm and 361 nm at 450 nm were chosen. From these, the following equations are valid10.
Rf = F361,450,Fura/F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 4 
RN1 = F400,500,NADH/F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 5 
RN2 = F353,500,NADH/F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 6
With these constants, the following equations from (Equation 1) (Equation 2), and (Equation 3) are valid.
F361,450 = F361,450,NADH + Rf &#215; F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 7 
F353,450 = RN2 &#215; F361,450,NADH + F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 8 
F400,500 = RN1 &#215; F361,450,NADH + F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 9
From these equations, if Rf, RN1, and RN2 are known, pure signals of NADH and fura-2 can be obtained as follows.
F361,450,NADH = (F361,450 - Rf &#215; F353,500)/(1 &#8722; Rf &#215; RN2) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 10 
F353,500,Fura = (RN2 &#215; F361,450 &#8722; F353,500)/(Rf &#215; RN2 &#8722; 1) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 11 
F400,500,Fura = F400,500 &#8722; RN1 &#215; F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; Equation 12 
RFura = F353,500,Fura/F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 13
The Ca2+-bound form of fura-2-FF was practically non-fluorescent at the 400 nm excitation wavelength. Based on this property, the following new calibration equation can be derived.
[Ca2+] = Kd &#8729; (F400,500,max/F353,500,max) &#215; (RFura &#8722; Rmin) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 14
where Kd is a dissociation constant, F400,500,max and F353,500,max are the maximum values of the emitted signals at 500 nm with excitations at 400 nm and 353 nm, respectively, and Rmin is the minimum RFura in Ca2+-free condition. Since the isosbestic excitations were used, the equation can be simplified further as follows.
[Ca2+] = Kd &#8729; (1 / Rmin) &#8729; (RFura &#8722; Rmin) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 15
Therefore, only Kd and Rmin values are required to calculate [Ca2+].

<table>
	<tbody>
		<tr>
			<td>2 mL eppendorf tube</td>
			<td>Axygen</td>
			<td>MCT-200-C</td>
			<td>2 mL Tube</td>
		</tr>
		<tr>
			<td>AD/DA converter</td>
			<td>Instrutech</td>
			<td>ITC-18</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>ADP, Adenosine 5&#8242;-diphosphate monopotassium salt dihydrate</td>
			<td>Sigma-aldrich</td>
			<td>A5285</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Band pass filter</td>
			<td>Ealing Electro-Optics, Inc</td>
			<td>35-3920</td>
			<td>Equipment, 640&#177;11nm</td>
		</tr>
		<tr>
			<td>Band pass filter</td>
			<td>Omega Optical</td>
			<td>690-9823</td>
			<td>Equipment, 590&#177;15nm</td>
		</tr>
		<tr>
			<td>Band pass filter</td>
			<td>Omega Optical</td>
			<td>500DF20-9916</td>
			<td>Equipment, 500&#177;20nm</td>
		</tr>
		<tr>
			<td>Band pass filter</td>
			<td>Chroma Technology Corp.</td>
			<td>60685</td>
			<td>Equipment, 450&#177;30nm</td>
		</tr>
		<tr>
			<td>Calcium chloride solution</td>
			<td>Sigma-aldrich</td>
			<td>21114</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>carboxy-SNARF-1(AM)</td>
			<td>Invitrogen</td>
			<td>C1272</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Charge-coupled device (CCD) camera</td>
			<td>Philips</td>
			<td>FTM1800NH/HGI</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>86009</td>
			<td>Equipment, Multiband dichroic mirror, Reflection : &#60;400nm, 490&#177;10, 560&#177;10, Transmission : 460&#177;15, 510&#177;20, &#62;580nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>567DCXRU</td>
			<td>Equipment, Reflection : &#60;560nm, Transmission : &#62; 580 nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>480dclp</td>
			<td>Equipment, Reflection : &#60;470nm, Transmission : &#62; 490 nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>20728</td>
			<td>Equipment, Multiband dichroic mirror, Reflection : &#60;405nm, 470&#177;30, Transmission : 430nm~520nm, &#62; 640 nm</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>Sigma-aldrich</td>
			<td>154938</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>DMEM, Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Sigma-aldrich</td>
			<td>D5030</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>EGTA, Egtazic acid, Ethylene-bis(oxyethylenenitrilo)tetraacetic acid, Glycol ether diamine tetraacetic acid</td>
			<td>Sigma-aldrich</td>
			<td>E4378</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>FCCP, Mesoxalonitrile 4-trifluoromethoxyphenylhydrazone</td>
			<td>Sigma-aldrich</td>
			<td>21857</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>field diaphragm</td>
			<td>Nikon</td>
			<td>86506</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Fura-2-FF(AM)</td>
			<td>TEFLABS</td>
			<td>137</td>
			<td>chemicals</td>
		</tr>
		<tr>
			<td>Green tube</td>
			<td>DWM</td>
			<td></td>
			<td>test tube</td>
		</tr>
		<tr>
			<td>HEPES,&#160; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid)</td>
			<td>Sigma-aldrich</td>
			<td>H3375</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>High-speed counter</td>
			<td>National Instruments</td>
			<td>NI-6022</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Hot mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>21002</td>
			<td>Equipment, 50:50</td>
		</tr>
		<tr>
			<td>Inverted microscope </td>
			<td>Nikon</td>
			<td>TE-300</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma-aldrich</td>
			<td>27606</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Near infrared filter </td>
			<td>Chroma Technology Corp.</td>
			<td>D750/100X</td>
			<td>Equipment, 750&#177;100nm</td>
		</tr>
		<tr>
			<td>Oil immersion lens </td>
			<td>Nikon</td>
			<td>MRF01400</td>
			<td>40x, NA 1.3; Equipment</td>
		</tr>
		<tr>
			<td>Photon counter unit</td>
			<td>Hamamatsu</td>
			<td>C3866</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Photon multiplier tube</td>
			<td>Hamamatsu</td>
			<td>R2949</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Polychrome II</td>
			<td>Till Photonics</td>
			<td>SA3/MG04</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>1.04936</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Potassium hydroxide solution</td>
			<td>Sigma-aldrich</td>
			<td>P4494</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-aldrich</td>
			<td>107360</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-aldrich</td>
			<td>R8875</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-aldrich</td>
			<td>S4521</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>TMRE, Tetramethylrhodamine, ethyl ester </td>
			<td>Molecular probes</td>
			<td>T669</td>
			<td>Chemicals</td>
		</tr>
	</tbody>
</table>

a novel nicotinamide adenine dinucleotide correction method for intracellular ca2+ measurement with fura-2-analog in live cells

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in live cells because of autofluorescence interference, mainly from nicotinamide adenine dinucleotide (NADH). More specifically, this is a major obstacle when measuring the mitochondrial [Ca2+] quantitatively using fura-2 analogs because the majority of NADH is in the mitochondria. If the fluorescent dye concentration is the same, a certain excitation intensity should produce the same emission intensity. Therefore, the emission intensity ratio of two different excitation wavelengths should be constant. Based on this principle, a novel online correction method of NADH signal interference to measure [Ca2+] was developed, and the real signal intensity of NADH and fura-2 can be obtained. Further, a novel equation to calculate [Ca2+] was developed with isosbestic excitation or excitation at 400 nm. With this method, changes in mitochondrial [Ca2+] could be successfully measured. In addition, with a different set of the excitation and emission wavelengths, multiple parameters, including NADH, [Ca2+], and pH or mitochondrial membrane potential (&#936;m), could be simultaneously measured. Mitochondrial [Ca2+] and &#936;m or pH were measured using fura-2-FF and tetramethylrhodamine ethyl ester (TMRE) or carboxy-seminaphtorhodafluor-1 (carboxy-SNARF-1).

Mitochondrial Ca2+ changes due to correction10 
Figure 4 shows the changes in [Ca2+]m before and after the correction. The results clearly showed the substantial changes in [Ca2+]m. The mitochondrial resting calcium concentration without cytosolic Ca2+ ([Ca2+]c) was 1.03 &#177; 0.13 &#181;M (mean &#177; S.E., n = 32), and the maximum [Ca2+]m at 1-&#181;M [Ca2+</s...

Watch this Scientific Journal Video about A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells at JoVE.com

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

Due to the spectral overlapping of the excitation and emission wavelengths of NADH and fura-2 analogs, the signal interference from both chemicals in live cells is unavoidable during quantitative measurement of [Ca2+]. Thus, a novel online correction method of NADH signal interference to measure [Ca2+] was developed.

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in ...

UV excitable plural probes, have an inherent signal contamination, because of the auto-plural sounds. Most of the problem with NADH, is limiting the use of a plural probe. A method can be used, to precisely correct the outer florescence from NADH, in order to extract a true, uncontaminated plural probe signal.Demonstrating the procedure will be Jeong Hoon Lee, our research assistant professor from my laboratory. After allowing the myocytes to settle to the bottom of the tube, aspirate the supernatant. Label the cells with two milliliters of freshly prepared one millimolar fura-2-FFAM diluting solution for 60 minutes at four degrees Celsius.At the end of the incubation, place the tube in a 37 degree Celsius water bath for 30 minutes in the upright position before removing the supernatant. Then add four milliliters of room temperature culture medium for storage at room temperature. For in situ isobestic fura-2-ffa point identification mount the diluted cells onto the microscope stage and wait three minutes for the cells to sink to the bottom.Profuse NADH-free 37 degree Celsius calcium-free solution into the cell culture and locate a target cell. Profuse saponin solution for 60 seconds before re-profusing with the NADH-free calcium-free solution. Simultaneously measure the fura-2-ff emitted signals at 450 and 500 nanometers, using an excitation scan from 350 to 365 nanometers with a 0.1 nanometer step and re-profuse with NADH-free calcium-free solution.Next subtract the signals in the calcium saturated solution from the signals in the calcium free conditions. Then calculate the standard deviations of the emission at each excitation and select the excitation wavelength showing the minimum standard deviation values as individual isobestic points. To use a multi-parametric measurement system to detect the background signal and to correct the signal within the cell area, mount dye-free cells in the microscope bath and allow the cells to settle for three minutes.Profuse NADH calcium-free solution into the bath for about five minutes at a two to three milliliters per minute rate and set the object field to cover the targeted cell. After moving the cell out of the field measure the background signals of the cell-free window and set the signals as offsets. Then return the cell to the initial position and measure the cell background signals and the cell area.To calculate the R-factor use the acquired cell background and area signals before remounting the dye-free cells onto the microscope and profusing with calcium-free solution. Measure the signals as indicated before profusing the cell suspension with malate solution. After measuring the signals profuse the cells with pyruvate solution followed by profusion with malate-pyruvate solution and rotenone solution.Then calculate the slope for each signal. Here the changes in mitochondrial calcium before and after the correction are shown. The results clearly reveal substantial changes in the mitochondrial calcium with a mitochondrial resting calcium concentration without cytosolic calcium of 1.03 plus or minus 0.13 micromolar and maximum mitochondrial calcium at one micromolar of calcium of 29.6 plus or minus 1.61 micromolar.The mitochondrial transmembrane potential was monitored with a profusion of two nanomolars TMRE. The change in mitochondrial potential was calculated based on an assumed initial mitochondrial membrane potential of minus 150 millivolts, demonstrating a decrease in NADH in response to calcium addition, with a negliable effect on the mitochondrial membrane potential. The mitochondiral pH was not effected by the increase in mitochondrial calcium induced by carboxy SNARF-1 loading.Incorrect isobestic points result in incorrect R and NADH correction. So take care to always correctly measure the cell-free background and cell area. This technique is used to prepared mitochondrial calcium dynamic studies in the bioanalytical spills.

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Research

JoVE Journal

Biology

6.6K vistas.  University of Ulsan College of Medicine/Asan Medical Center. Sondas plurales excitables UV, tienen una contaminación de señal inherente, debido a los sonidos auto-plurales. La mayor parte del problema con NADH, está limitando el uso de una sonda plural. Se puede utilizar un método para corregir con precisión la florescencia externa de NADH, con el fin de extraer una señal de sonda plural verdadera y no contaminada.Demostrando el procedimiento estará Jeong Hoon Lee, nuestro profesor asistente de investigación de mi laboratorio. Después d...