Name: use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model
Uploaded: 2014-04-16T15:25:00
Description: Watch this Scientific Journal Video about Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model at JoVE.com

这里所描述的工作是由退伍军人事务部，美国能源部生物医学实验室的研究和开发BX001686-1A1和VA康复研究和开发。

1。电镀细胞培养和维护<ol><li>温暖的Dulbecco改良的Eagle培养基（DMEM）培养基（DMEM +10％FBS +1％青霉素/链霉素/新霉素）至37℃。 </li><li>获得冷冻A12细胞的股价从-80°C。 </li><li>快速解冻细胞37℃水浴;一旦解冻，轻轻吸细胞转移到一个75 厘米通风培养瓶中，并加入10 ml培养基。 </li><li>烧瓶孵育过夜，5％CO 2培养箱在37°C吸介质后24小时，更换新鲜的培养基。持续到70-80％汇合得到（2-3天...

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在多重检测是一个已经在许多应用，如PCR芯片，免疫检测，以及其他基于蛋白质的检测方法13，14被科学家广泛接受的技术。最近，复用实验已经越来越利用体外板基础实验和已被确认为评估细胞毒性的准确方法和可行性12。在上述协议中，我们证明了复用荧光灯和发光检测的有效性，以确定在A12单元格下面的PA挑战的caspase-3/7活性和细胞活力。细胞活力，在初始的PA侮辱10分?...

饮食富含饱和脂肪酸如棕榈酸（PA）的已与肥胖和其他并发症，包括心血管疾病和糖尿病1,2。高脂肪饮食也已显示出增加的氧化应激，细胞凋亡，和在下丘脑的神经元变性，食欲和能量消耗3-7中的重要调节器。了解通过高脂饮食诱导的曝光下丘脑功能失调的机制是对肥胖的药物治疗发展重要的。然而，通过这些食物中的脂肪会影响神经元功能的细胞机制仍不清楚。更好地理解如何脂肪如PA可能会引发发病丘脑凋亡途径是迈向这一目标的必要的第一步。本文的目的是描述一个多重检测用于体外测试与PA曝光神经元的反应，对于在H的研究开发利用ypothalamic神经退行性病变。我们提供了一种体外的96孔格式的多重检测，用于测量胱天蛋白酶每个细胞中分化的永生化的成体小鼠的下丘脑细胞与PA的8氧化攻击后线（称为A12）总数3/7活性的详细说明。 简单地说，细胞存活率确定通过基于刃天青分析如下PA的挑战。刃天青是经历酶减少，代谢活跃的细胞的细胞渗透性化合物想到一个过程，通过线粒体9出现。活细胞连续转换刃天青到试卤灵，产生的荧光信号正比于活细胞的数量。胱天蛋白酶3/7活性，随后分析使用DEVD-基于lumogenic测定。 DEVD是由胱天蛋白酶-3切割的氨基酸序列（ASP-GLU-VAL-ASP）。当该序列被耦合到lumogenic物质，在激活时细胞内的胱天蛋白酶3/7的和随后的裂解DEVD基板，发光产物被释放。该反应正比于胱天蛋白酶活性，从而诱导细胞凋亡。由于死细胞不能产生，即caspase-3/7活性本质上是短暂的;因此分析应该在30分钟至4小时后挑战之间完成，这取决于细胞应激的效果。细胞活力是成反比的胱天蛋白酶3/7活性，并且可以用于确定细胞死亡的机制。例如，该方法以前已经用来表明预处理与肽激素食欲减少细胞凋亡的挑战与过氧化氢 ​​的下丘脑细胞，这表明影响此治疗机制在防止氧化损伤10重要。要注意这些试验是细胞系和组织依赖性的，因为它们依赖于线粒体活性降低测定试剂是非常重要的。该协议已被优化，成年小鼠的下丘脑（A12）细胞;然而，所描述的方法可以被改变，以适应类似研究的范围之内。 从一个单一的复用培养测定以及提供了对个体进行测定几个原因的传统方法的优点。除了 ​​节省时间，细胞样本，并培养试剂，复分析还可以提供细胞存活和死亡的知识，提供内部控制，并省去了重复实验，11，12。 可供选择的方法都依赖于蛋白质印迹或ELISA中，这是可靠的测定法，但价格昂贵和费时的（1-2天），使用96孔形式时尤其如此。不含花费来培养细胞为复测定的时间，总时间不超过3小时。虽然该协议已被优化，与A12单元格的使用，它可以同时考虑的因素可能影响细胞的完整性来改变用于其他车型。阻止如果该协议将工作的一系列实验采矿取决于样本或实验条件的数量以及规划下游实验。

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Adult Mouse Hypothalamus Cell Line mHypoA-1/2&#160;</td>
			<td style="width:137px;">Cellutions Biosystems Inc.</td>
			<td style="width:151px;">CLU172</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">10313-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Fetal bovine serum&#160;</td>
			<td style="width:137px;">PAA Labs</td>
			<td style="width:151px;">A15-751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Penicillin/Streptomycin</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Palmitic Acid</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dimethy sulfoxide&#160;</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;"> 
			PrestoBlue Cell Viability Reagent</td>
			<td style="width:137px;">Invitrogen</td>
			<td>A13262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Caspase-Glo 3/7 Assay Systems</td>
			<td style="width:137px;">Promega</td>
			<td style="width:151px;">G8091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Equipment</td>
			<td style="width:137px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:268px;">96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td>
			<td style="width:137px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">165305</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">SpectraMax M5 Multi-Mode Microplate</td>
			<td style="width:137px;">Molecular Devices</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model

复测定法中的复杂的细胞机制的研究的能力，省去了重复的实验中，提供了内部控制，并在成本和试剂减少浪费。这里，我们描述了多重测定，以评估细胞凋亡的优化以下棕榈酸（PA）的挑战， 在体外模型中的下丘脑，同时使用荧光和发光为基础的分析来测量在96孔的活细胞计数和caspase-3/7活性微量滴定板形式。下面的PA挑战，存活细胞用刃天青类荧光测定法测定。胱天蛋白酶3/7活性是再使用一个luminogenic基板，DEVD，并且标准化为细胞数目确定。此复用测定法是用于确定变化caspase活性以下的凋亡刺激物，如饱和脂肪酸挑战的有效技术。饱和脂肪酸的PA可以增加下丘脑的氧化应激和细胞凋亡，表明了潜在importa在研究饱和脂肪酸和神经元功能之间的关系描述此处测定法如的考。

上述协议描述的结果在复用两个独立的实验，以确定细胞死亡的机制。 图1显示了协议的概述，以确定细胞活力和caspase-3/7活性。半胱天冬酶活性的细胞后2小时温育（ 图2A和表1）挑战与PA显著增加。的细胞膜完整性的丧失与细胞凋亡的诱导，这是明显的细胞后2小时暴露到PA（ 图2B）相关的形态学变化， 图3概括了PA诱导细胞凋亡，并演...

Watch this Scientific Journal Video about Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model at JoVE.com

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

多重分析可以提供有益的信息为基础的细胞机制，消除试剂和不必要的重复实验的浪费。我们在这里描述的多路胱天蛋白酶3/7活性测定法，使用荧光和发光为基础的方法，以确定在以下与棕榈酸的氧化挑战的体外模型中的下丘脑细胞活力。

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

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