Name: high-resolution respirometry to assess mitochondrial function in permeabilized and intact cells
Uploaded: 2017-02-08T13:00:00
Description: Watch this Scientific Journal Video about High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells at JoVE.com

This study was supported by the Swiss National Science Foundation (Grant n&#186; 32003B_127619).

1. خلية ثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ثقافة الخلايا البشرية الكبدي HepG2 6 في 25 سم 2 قوارير زراعة الخلايا في المتوسط Dulbecco لتعديل النسر (DMEM) تحتوي على 10٪ للحرارة المعطل مصل بقري جنيني (FBS) و 1٪ البنسلين ستربتومايسين عند 37 درجة مئوي...

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وكان الهدف من هذا البروتوكول إلى استخدام عالية الدقة قياس التنفس لقياس المجمعات السلسلة التنفسية الميتوكوندريا &quot;(I-IV) وتيرة التنفس، أقصى قدرة نظام نقل الإلكترون الميتوكوندريا والميتوكوندريا سلامة الغشاء الخارجي. هناك بعض الخ...

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		<tr height="24">
			<td height="24" style="height:24px;width:156px;">ADP</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">A 4386</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:156px;">Antimycin A</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">A 8674</td>
			<td style="width:92px;">Chemical, dissolve in ethanol</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:156px;">Ascorbate</td>
			<td style="width:92px;">Merck</td>
			<td style="width:75px;">1.00127</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">BSA</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">A 6003</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">FCCP</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">C 2920</td>
			<td style="width:92px;">Chemical, dissolve in ethanol</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:156px;">Countess automated cell counter&#160;</td>
			<td style="width:92px;">Thermo Fisher Scientific</td>
			<td style="width:75px;">n/a</td>
			<td style="width:92px;">Automated cell counting instrument</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Cytochrome c</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">C 7752</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Digitonin</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">D 5628</td>
			<td style="width:92px;">Chemical, dissolve in DMSO</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">DMEM&#160;</td>
			<td style="width:92px;">Gibco</td>
			<td style="width:75px;">31966021</td>
			<td style="width:92px;">Medium</td>
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		<tr height="24">
			<td height="24" style="height:24px;width:156px;">EGTA</td>
			<td style="width:92px;">fluka</td>
			<td style="width:75px;">3779</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">FBS</td>
			<td style="width:92px;">Gibco</td>
			<td style="width:75px;">26010-074</td>
			<td style="width:92px;">Medium component</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Glutamate</td>
			<td style="width:92px;">Sigma,</td>
			<td style="width:75px;">G 1626</td>
			<td style="width:92px;">Chemical</td>
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		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Hepes</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">H 7523</td>
			<td style="width:92px;">Chemical</td>
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			<td height="24" style="height:24px;width:156px;">KCl</td>
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			<td style="width:75px;">1.04936</td>
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			<td height="24" style="height:24px;width:156px;">KH2PO4</td>
			<td style="width:92px;">Merck</td>
			<td style="width:75px;">1.04873</td>
			<td style="width:92px;">Chemical</td>
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		<tr height="24">
			<td height="24" style="height:24px;width:156px;">K-lactobionate</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">L 2398</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">MgCl2</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">M 9272</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:156px;">O2k-Core: Oxygraph-2k&#160;</td>
			<td style="width:92px;">Oroboros Instruments</td>
			<td style="width:75px;">10000-02</td>
			<td style="width:92px;">High-resolution respirometry instrument</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Oligomycin</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">O 4876</td>
			<td style="width:92px;">Chemical, dissolve in ethanol</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Penicillin-streptomycin</td>
			<td style="width:92px;">Gibco</td>
			<td style="width:75px;">15140122</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Sodium azide</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">S2002</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Rotenone</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">R 8875</td>
			<td style="width:92px;">Chemical, dissolve in ethanol</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Succinate</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">S 2378</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Taurine</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">T 8691</td>
			<td style="width:92px;">Chemical</td>
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		<tr height="24">
			<td height="24" style="height:24px;width:156px;">TMPD</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">T 3134</td>
			<td style="width:92px;">Chemical</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td style="width:92px;">Sigma</td>
			<td>T 4674</td>
			<td style="width:92px;">Chemical</td>
		</tr>
	</tbody>
</table>

high-resolution respirometry to assess mitochondrial function in permeabilized and intact cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

تحديد الأمثل ديجيتونين تركيز الخلوية Permeabilization: ديجيتونين المعايرة تجربة يتم تنفيذ ديجيتونين المعايرة لتحديد تركيز الأمثل لpermeabilization الخلايا HepG2. لهذه التجارب، ومعاير ديجيتونين في خلايا سليمة ف...

Watch this Scientific Journal Video about High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells at JoVE.com

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

يستخدم عالية الدقة قياس التنفس لتحديد استهلاك الأوكسجين الميتوكوندريا. هذا هو أسلوب واضحة لتحديد المجمعات السلسلة التنفسية الميتوكوندريا &quot;(I-IV) وتيرة التنفس، أقصى قدرة الميتوكوندريا نظام نقل الإلكترون، والميتوكوندريا سلامة الغشاء الخارجي.

عالية الدقة قياس التنفس لتقييم وظيفة الميتوكوندريا في Permeabilized وخلايا سليمة

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

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Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

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