Name: a novel nicotinamide adenine dinucleotide correction method for intracellular ca2+ measurement with fura-2-analog in live cells
Uploaded: 2019-09-20T14:00:00
Description: 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

这项工作部分得到韩国国家科学研究基金会（NRF）基础科学研究计划的支持，该基金会由韩国教育部资助（2018R1A6A3A0101011832），由科学、ICT和未来规划部（NRF-2016M3C1A693666）资助。贸易、工业和能源部 （10068076）。

所有实验协议都通过了当地机构动物护理和使用委员会的批准。
1. 解决方案准备
<ol><li>准备单新鲜分离的心脏肌细胞11。 注：每个实验室可能有不同的细胞存储解决方案。在这里，肌细胞存储在培养基（DMEM）中。</li>
	<li>准备 100 mL 的 Ca2+无解决方案 （表 1.</li>
	<li>在 50 mL 烧杯中准备 50 mL 的培养基。5 mL，在 37°C 下放入水浴中。将剩...

<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>

成功地开发了用于测量NADH和fura-2模拟信号的干扰校正方法。准确测量信号对于精确校正至关重要。然而，荧光装置的固有特性会产生与Fura-2的NADH无关的背景信号。最高质量的带通滤波器只能传递多达10~8个不需要的光波长。然而，来自单个细胞的荧光信号非常小，带通滤波器后激发光的反射仍然足够强，足以污染实际的荧光信号。因此，有必要仔细校正背景信号。
Fur...

细胞内Ca2+的重要作用是众所周知的1。[Ca2]的量化对于了解细胞生理功能的过程至关重要。Fura-2 类比非常有用，因为它们在 UV 范围（<400 nm）中激发，比例测量方法可用于定量测量。因此，其他生理参数，如pH、膜电位等，可以使用其他荧光染料进行测量。据报道，线粒体Ca2+浓度（[Ca2+]m）范围为0.08±20 μM2，3，4，5。在 fura-2 模拟中，fura-2-FF 适用于测量此范围 [Ca2]。然而，不幸的是，活细胞含有NADH/NADPH的代谢过程，NADH产生信号干扰，因为与fura-2模拟的激发和发射光谱重叠。这种干扰极大地限制了 fura-2 模拟物的使用。具体来说，如果应用模拟测量线粒体[Ca2]，这种干扰是最大的障碍，因为NADH的最大量是线粒体。这与线粒体膜电位 （μm）相关的 NADH 变化进一步复杂化，而 αm的变化影响 [Ca2]m6，7，8,9.此外，为了研究[Ca2]m动力学，必须了解其他线粒体参数（如NADH、μm和pH值）的状态。
450 nm 和 500 nm 的排放，在 353 nm、361 nm 和 400 nm 的激发下包含来自 NADH 和 fura-2-FF 的信号，方程如下。在这里，353 nm 和 361 nm 是 fura-2-FF 的等位，分别用于 450 nm 和 500 nm 的排放。
F361，450 = F361，450，NADH = F361，450，富拉方程 1 F353，500 = F353，500，NADH = F353，500，富拉方程 2 F400，500 = F400，500，NADH = F400，500，Fura方程 3
其中 Fx，y是通过 x-nm 激发在 y-nm 处测量的发射强度，F x，y，NADH表示纯 NADH 相关发射强度，而 Fx，y，Fura表示纯 fura-2-FF 依赖性发射强度。在荧光染料的相同浓度下，一定的激发强度应产生相同的发射强度。因此，两个不同激发波长的发射强度比应该是恒定的。Ca2+和 fura-2 不影响 NADH 荧光特性;因此，在450nm和500nm的NADH下，在任何激发波长下，发射率是恒定的。基于NADH或[Ca2]不影响fura-2-FF的发射和激发光谱的假设，同样的规则可用于fura-2-FF。但是，Ca2+导致 fura-2-FF 发射的光谱偏移。因此，要消除Ca2+的影响，需要使用独立于Ca2+的等位激励。每个发射波长（即 450 nm 和 500 nm）具有不同的异位，从我们的实验设置中，选择 353 nm 在 500 nm，361 nm 在 450 nm。从这些，以下方程是有效的10。
Rf = F361，450，富拉/F353，500，富拉方程 4 RN1 = F400，500，NADH/F361，450，NADH方程 5 RN2 = F353，500，NADH/F361，450，NADH方程 6
对于这些常数，以下（公式 1）（方程 2）和（方程 3）的方程有效。
F361，450 = F361，450，NADH = Rf = F353，500，富拉方程 7 F353，450 = RN2 = F361，450，NADH = F353，500，富拉方程 8 F400，500 = RN1 = F361，450，NADH = F400，500，富拉方程 9
从这些方程中，如果已知Rf、RN1和RN2，则可以获得NADH和fura-2的纯信号，如下所示。
F361，450，NADH = （F361，450 - Rf = F353，500）/（1 = Rf = RN2）方程 10 F353，500，Fura = （RN2 = F361，450 = F353，500）/（Rf = RN2 = 1） 方程 11 F400，500，Fura = F400，500 = RN1 = F361，450，NADH方程 12 RFura = F353，500，富拉/F400，500，富拉方程 13
在400nm激发波长下，Fura-2-FF的Ca2+结合形式几乎是非荧光的。基于此属性，可以派生出以下新的校准方程。
[Ca2]= = Kd = （F400，500，最大/F353，500， 最大值） = （RFura = R分钟）方程 14
其中 Kd是解散常数，F400，500、max和 F353，500，最大值是 500 nm 时发射信号的最大值，激发分别为 400 nm 和 353 nm，Rmin是 Ca 2 中的最低 RFura *-免费条件。由于使用了等位激励，可以进一步简化方程，如下所示。
[Ca2+= Kd = （1 / R分钟）] （RFura = R分钟）方程 15
因此，只需 Kd和 R最小值即可计算 [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

为了定量测量[Ca2]，经常使用fura-2类比，即比例荧光探针。然而，由于自荧光干扰，染料在活细胞中的使用受到内在限制，主要来自烟酰胺腺苷二核苷酸（NADH）。更具体地说，这是使用 fura-2 模拟体定量测量线粒体 [Ca2]时的一个主要障碍，因为 NADH 的大部分位于线粒体中。如果荧光染料浓度相同，则一定的激发强度应产生相同的发射强度。因此，两个不同激发波长的发射强度比应该是恒定的。基于此原理，开发了一种新型的NADH信号干扰测量在线校正方法，并得到了NADH和fura-2的实际信号强度。此外，在400nm处，在无共态激发或激发下，开发了一种计算[Ca2]的新方程。使用这种方法，可以成功地测量线粒体 [Ca2]的变化。此外，使用一组不同的激发和发射波长，可以同时测量多个参数，包括NADH、[Ca2]和pH或线粒体膜电位（μm）。线粒体 [Ca2]和μm或 pH 值使用 fura-2-FF 和四甲基二甲胺乙酯 （TMRE） 或半磷酸二酯-1（卡博比-SNARF-1）进行测量。

线粒体 Ca2+因校正10而改变 图 4显示了更正前后[Ca2]m 中的更改。结果清楚地表明了[Ca2]m的重大变化。无细胞Ca2+（[Ca 2+]c ） 的线粒体静钙浓度为 1.03 ± 0.13 μM （均值 = S.E.， n = 32），最大 [Ca2]m在1-μM [Ca2]c为 29.6 ± 1.61 μM （均值 = S.E.，n = 33） （图 5）。...

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

由于NADH和fura-2类似物的激发和发射波长的光谱重叠，在[Ca2]的定量测量中，活细胞中两种化学物质的信号干扰是不可避免的。因此，开发了一种新型的NADH信号干扰在线校正方法，以测量[Ca2]。

一种新型的尼古丁酰胺二丁二核苷酸校正方法，用于在活细胞中使用Fura-2-模拟进行细胞内Ca2+测量

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

UV兴奋复数探头，由于自动复数声音，具有固有的信号污染。NADH 的主要问题是限制复数探头的使用。一种方法，可以精确地从NADH的外荧光，以提取一个真正的，未污染的复数探头信号。演示这个程序的将是我实验室的研究助理教授李俊俊。在允许菌分泌到管底后，吸进上清液。用两毫升新鲜准备的一毫升毛茸茸-2-FFAM稀释溶液在4摄氏度下为细胞贴上60分钟的标签。在孵育结束时，将管子放在37摄氏度的水浴中，在直立位置放置30分钟，然后取出上经器。然后加入四毫升的室温培养介质，在室温下储存。对于原位是肥胖的毛茸茸-2-ffa点识别将稀释的细胞安装到显微镜阶段，并等待三分钟，让细胞沉入底部。将无 NADH 无钙溶液移植到细胞培养中并定位目标细胞。使用无 NADH 无钙溶液再使用皂素溶液 60 秒。同时测量 450 和 500 纳米的 fura-2-ff 发射信号，使用 350 到 365 纳米的激励扫描，步长为 0.1 纳米，并使用无 NADH 无钙溶液进行再 prof。接下来从无钙条件下的信号中减去钙饱和溶液中的信号。然后计算每个激发时发射的标准偏差，并选择显示最小标准差值作为单个肥胖点的激励波长。要使用多参数测量系统来检测背景信号并纠正细胞区域内的信号，请将无染料细胞安装在显微镜浴中，并允许细胞沉淀三分钟。将 NADH 无钙溶液以每分钟两到三毫升的速度放入浴缸中约五分钟，并设置对象场以覆盖目标细胞。将单元格移动到字段后，测量无单元格窗口的背景信号，并设置信号为偏移。然后将单元格返回到初始位置并测量单元格背景信号和单元格区域。要计算 R 因子，请使用获得的细胞背景和面积信号，然后再将无染料细胞重新安装到显微镜上，并使用无钙溶液进行补充。在用麦芽溶液使细胞悬浮液扩散之前，测量指示的信号。测量信号后，用丙酮酸盐溶液大量测量细胞，然后用麦芽酸-丙酮溶液和罗泰诺溶液大量注入。然后计算每个信号的斜率。这里显示纠正之前和之后线粒体钙的变化。结果清楚地显示线粒体钙的显著变化，线粒体休息钙浓度没有细胞酸钙1.03加或减0.13微摩尔和最大线粒体钙在一微摩尔钙29.6加或负1.61微摩尔。线粒体跨膜电位监测与两个纳米摩尔TMR的大量监测。线粒体电位的变化是根据假定的初始线粒体膜电位为负150毫伏计算出来的，表明NADH对钙添加的反应减少，对线粒体膜电位有不可忽略的影响。线粒体pHH没有受到碳氧SNARF-1负荷引起的线粒体钙增加的影响。不正确的是肥胖点会导致不正确的 R 和 NADH 校正。因此，请注意始终正确测量无细胞背景和单元格区域。该技术用于在生物分析溢出中准备线粒体钙动态研究。

Research

JoVE Journal

Biology

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

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科研

生物学

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