作者承认放射生物学服务（里昂 - 萨德医院）的安妮·查里（AnneCharrié）以及Fond National Fis（FNS）的财务支持。

1.细胞培养基和溶液的制备<ol><li> 培养基的制备 <ol><li>通过补充Ham&#39;s F-10培养基与谷氨酰胺（2mM），青霉素/链霉素（最终5μg/ mL），2％胎牛血清（FCS）和2％血清替代物来制备增殖培养基（PM）。 </li><li>通过用谷氨酰胺（2mM），青霉素/链霉素（最终5μg/ mL）和2％FCS补充Dulbecco&#39;s Modified Eagle培养基（DMEM）来制备分化培养基（DM）。 </li></ol></li><li> 葡萄糖摄取溶液的制备 注意:授权人员只能在受限制的区域内处理放射性物质。材料和废物必须按照适当的程序，指导原则和当地立法处理。 <ol><li>为了制备X-Dulbecco的磷酸盐缓冲盐水（X-DPBS），制备含有0.2％（w / v）（终浓度）牛血清白蛋白umin（BSA）。通过0.2μm过滤器过滤溶液。储存于4°C。 </li><li>为了制备冷的2-脱氧-D-葡萄糖（2dG）溶液，称重16.4mg的2dG并溶解在10mL蒸馏水中以获得10mM溶液。储存于4°C。 </li><li>加入600μL冷2dG和6μL放射性标记的[ 3 H] 2dG至5400μLX-DPBS，得到放射性标记的2dG（2dG *）溶液。 注意:最终浓度为1 mM 2dG，标记为1μCi/ mL。 <ol><li>放置放射性标记的2dG *溶液的20μL等分试样（TC20）。 </li></ol></li></ol></li><li> 孵育混合物的制备 <ol><li>对于细胞松弛素B混合物，加入2μL20mM细胞松弛素B至2mL放射性标记的2dG *溶液。 注意:细胞松弛素B的储备溶液在二甲基亚砜（DMSO）中为10mg / mL。 </li><li>对于DMSO混合物，向剩余的4mL放射性标记的2dG *溶液中加入4μLDMSO。 </li></OL&gt; </li></ol> 2.人原代肌细胞培养<ol><li> 用人类肌肉卫星细胞播种6孔板 注意:从冷冻小瓶（含有25万个细胞）内部使用（详见参考资料24 ）或市售的人类肌肉卫星细胞。给出250,000个细胞的以下程序，以获得在单一条件下测量葡萄糖摄取所必需的一个6-孔板。 <ol><li>在预热的水（37℃）中快速解冻冷冻的小瓶内部的24或人类肌肉卫星细胞的商业制剂，直到小瓶中只剩下一小块冰块。 </li><li>直接倒入含有10mL预热（37℃）PM的50mL塑料管中。 </li><li>以500×g离心5分钟，弃去上清液。 </li><li>用18mL预热的PM轻轻重悬细胞沉淀（每个获得42,000个细胞）3 mL培养基）。在6孔板（9.6cm 2 ）的每个孔中分配3mL。 注意:对于以下条件，板的六个单独的孔需要进行葡萄糖摄取的重复测量:被动运输抑制（孔1和2），基础速率（孔3和4）和胰岛素刺激速率（孔5和6）。重复几个六孔板需要不同的处理。 </li><li>在标准培养条件（37℃，5％CO 2 ）中孵育，直到细胞达到90％汇合。 注意:根据细胞批次，此步骤需要48 - 72 h。在此步骤中不要更换介质。 </li></ol></li><li> 肌肉细胞分化 <ol><li>去除PM（48-72小时后），并用预热的DM（3mL /孔）代替。在37℃，5％CO 2下孵育。 注意:分化需要五天才能达到细胞排列和多核的稳定状态。通常，主要肌管在1g / L葡萄糖培养基中培养。因此，为了避免在培养过程中葡萄糖消耗，用3 mL培养基填充平板，以确保足够的葡萄糖底物在任何时间都可用于细胞。 </li><li>每两天更换DM培养基。 注意:从这一点来看，肌管长达7天是稳定的，没有任何显着的变化，并且可以随时进行葡萄糖摄取测量。 </li></ol></li><li> 肌肉细胞治疗（可选） 注意:在胰岛素刺激和葡萄糖摄取测量之前，主要肌管可以治疗数天以诱导修饰（药物测试，信号通路的抑制剂/激活剂等 ）。肌肉细胞可以进行任何可能对胰岛素敏感性影响的治疗，葡萄糖摄入测量将量化这种影响。例如，肌肉细胞与饱和脂肪酸棕榈酸酯的孵育促进胰岛素抵抗，细胞显示减少i苏木精刺激葡萄糖摄取。 <ol><li>准备12 mL补充有10％BSA（不含脂肪酸）和0.5 mL棕榈酸酯（PALM）的DM。准备12 mL补充有10％BSA（不含脂肪酸）的DM。 </li><li>用人原代肌管制备两个6孔板，按2.1和2.2节（分化5天）所述进行培养。 </li><li>在第5天，用2mL PBS洗涤每个孔。向一个平板中加入2mL含有PALM的DM。向其他板中加入2mL仅含有DM的BSA。 </li><li>在37℃，5％CO 2下孵育48小时。 </li></ol></li></ol> 3.胰岛素刺激<ol><li>用2mL PBS洗涤分化的肌肉细胞两次。 </li><li>仔细取出PBS，并用3 mL无FCS的DM孵育3 h（37°C，5％CO 2 ）用于血清消耗。 </li><li>用不含FCS的3mL DM替换所有孔中的培养基。向孔5和6添加100nM胰岛素。 </li><li>孵育人肌管培养1小时（37℃，5％CO 2 ）。 </li></ol>葡萄糖摄取<ol><li>胰岛素刺激1小时后，用X-DPBS洗涤两次（每次洗一毫升）。 </li><li>将1mL细胞松弛素B混合物加入孔1和2，加入1mL DMSO混合物至孔3-6。孵育15分钟（37℃，5％CO 2 ）。孵化结束后，立即将板放在冰上。 </li></ol>细胞裂解<ol><li>用1 mL冰冷的PBS清洗细胞两次。 </li><li>用600μL的50mM NaOH溶解各孔中的细胞。在冰上孵育5分钟，并慢慢地与轨道旋转混合。 注意:如果裂解物太粘稠，最多可用1.5 mL NaOH稀释。 </li><li>使用移液管，重悬和收集细胞裂解液。 </li></ol> 6.放射性标记葡萄糖的测定<ol><li>将每个细胞裂解物400μL放入液体闪烁计数小瓶中。准备一个400的阴性对照小瓶μL50mM NaOH和20μLTC20的阳性对照小瓶（来自步骤1.2.3.1）。 </li><li>向每个小瓶中加入4 mL液体闪烁溶液。关闭盖子并彻底混合每个小瓶（1-2秒）。 </li><li>将每个小瓶插入液体闪烁计数器，并根据制造商的说明测量放射性。每个闪烁瓶记录每分钟的计数（CPM）10分钟。 注意:CPM ="每分钟分解"x"计数效率"。 </li></ol> 7.葡萄糖摄取率<ol><li>使用剩余的裂解物（200μL;来自步骤5.2）来测量蛋白质浓度。使用Bradford 25或等效方法确定每个细胞裂解物的蛋白质浓度。计算每个孔的总蛋白质量（Q）（mg）。 </li><li>要获得TC1（1μL放射性标记的2dG *的值），将TC20的CPM值除以20。 </li><li>对于每个小瓶，计算t他的葡萄糖摄取率如下: <img alt="方程" src="/files/ftp_upload/55743/55743eq1.jpg" /> 注意:R以pmol / mg / min测量。井1-2的平均值为被动运输速率，R p 。井3-4的平均值为基础总运输率，R bt 。孔5-​​6的R平均值给出胰岛素刺激的总运输速率R。 <ol><li>计算基础活动运输率（R ba ）如下: <img alt="方程" src="/files/ftp_upload/55743/55743eq2.jpg" /></li><li>计算胰岛素刺激的活动转运率（R ia ）如下: <img alt="方程" src="/files/ftp_upload/55743/55743eq3.jpg" /> 注意:在胰岛素反应性细胞如肌管中，葡萄糖摄取率通常由三个值表示:R ba ，R ia和胰岛素刺激倍数为R ia / R ba 。 </li></ol></li></ol>

作者没有什么可以披露的。

葡萄糖摄取是测试细胞培养上的活化剂或抑制剂以及它们如何影响葡萄糖使用以及细胞对胰岛素反应的能力的关键生物学测量。已经显示这里描述的方法是快速和可靠的，并已被广泛应用于使用来自健康受试者和/或代谢影响患者6,7,10,12,21,26,27,36,37的主要肌管的许多研究。只有一个6孔板，可以获得总基础运输，基础活动运输和胰岛素刺激主动运输的重复率。这三个值完全描述了体外培养的?...

骨骼肌是哺乳动物中最大的葡萄糖沉积物，在很大程度上有助于葡萄糖的体内平衡。这种胰岛素反应性组织是由胰岛素刺激引起的葡萄糖摄取的主要部位1 。 在2型糖尿病中，在包括骨骼肌在内的几种组织中观察到胰岛素抵抗，并导致高于正常血糖浓度。因此，确定该组织及其细胞的胰岛素敏感性的水平是重要的，无论目标是表征受试者的缺陷，还是评估旨在改善其的治疗效率。在人或动物受试者中，评估胰岛素敏感性的金标准技术是高胰岛素血症 - 正义血糖钳夹。由DeFronzo于1979年推出，并自3月 4日修改，然后，该方法允许量化全身and组织胰岛素反应性测量为在胰岛素刺激下灌注的葡萄糖的速率以维持正常的血糖浓度。 也可以使用体外肌肉模型在细胞水平进行胰岛素敏感性检测，并且葡萄糖摄取率的测量仍然是量化细胞对胰岛素刺激的生物反应的有效和可靠的工具5,6,7 。实际上，葡萄糖摄取测量量化胰岛素刺激的细胞生物学反应，从胰岛素与其受体结合到富含GLUT4的囊泡的易位，并包括细胞内信号传导和磷酸化级联8 。 这是人类样本的主要兴趣，因为分化的肌管维持供体表型的许多特征，包括代谢性质在患者9,10,11,12中观察到的病症和病症。肌管显示与骨骼肌13,14的结构，代谢和表型相似性，包括葡萄糖转运蛋白15和细胞胰岛素信号传导机制16的表达 。因此，初级肌管中葡萄糖摄取的测量与表征供体的肌肉表型相关，或调查干预（药物，营养或身体活动）对肌肉细胞胰岛素敏感性的影响。 在进行修改胰岛素敏感性17,18的实验时，培养的肌管上葡萄糖摄取的测量也是可靠的工具。 体外 </em&gt;模型适用于可以改善胰岛素反应性的任何化合物的测试，或可以预防或逆转所获得或诱导的胰岛素抵抗19,20,21,22,23 。 在这里我们描述一个详细的培养和区分人肌管的方案，并测量细胞葡萄糖摄取率。该方法适用于人类肌肉前体细胞的任何来源，无论它们来自实验室内的制备，合作或商业供应商。分别来自小鼠和大鼠来源的永生化肌肉细胞系（如C2C12和L6）也可用于本方案的葡萄糖摄取测定。 我们提供了一个详细的方案来定量使用放射性标记的[ 3 H] 2dG的基础和胰岛素刺激状态的速率。 Ť他使用标记的葡萄糖类似物可以准确测定葡萄糖进入与起始原料减少，这是当与原代细胞一起工作时的常见情况。修饰的葡萄糖分子不能进入代谢途径，因此积累在细胞内，允许通过总细胞放射性进行可靠的定量。实验条件包括使用葡​​萄糖转运抑制剂（细胞松弛素B），并且使用和不使用胰岛素进行测量。该组合允许测定葡萄糖活性进入率，以及计算胰岛素反应指数的倍数变化。该方法在单个孵育时间内呈现一剂胰岛素，但是该方案可以容易地修改用于剂量反应或时间过程实验12 。

<table border="0" cellpadding="0" cellspacing="0" width="868">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Human primary muscle cell</td>
			<td style="width:171px;">In house preparation from human skeletal muscle biopsies</td>
			<td style="width:180px;">In house preparation from human skeletal muscle biopsies</td>
			<td style="width:257px;">If not available, use commercial source</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Human primary muscle cell</td>
			<td style="width:171px;">Promocell</td>
			<td>C-12530</td>
			<td style="width:257px;">Should be cultured with associated media C23060 and C23061</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">6-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:180px;">356400</td>
			<td style="width:257px;">BioCoat Collagen I Multiwell Plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Ham&#39;s F10</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0145-500</td>
			<td style="width:257px;">1 g/l glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Glutamine</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">X0551-100</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">penicilin/streptomycin 100x</td>
			<td style="width:171px;">Thermo fisher scientific</td>
			<td style="width:180px;">15140122</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Serum substitute UltroserG</td>
			<td style="width:171px;">Pall France</td>
			<td style="width:180px;">15950.017</td>
			<td style="width:257px;">serum substitute in text</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">DMEM low glucose</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0064-500</td>
			<td style="width:257px;">1 g/l glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Fetal Calf Serum</td>
			<td style="width:171px;">Eurobio</td>
			<td style="width:180px;">CVFSVF00-01</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0625-500</td>
			<td style="width:257px;">Contains Mg2+ (0.5 mM) and Ca2+ (0.9 mM)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Insulin solution human</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">I9278</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">2-deoxy-D-glucose&#160;</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">D6134</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Albumin bovine</td>
			<td style="width:171px;">euromedex</td>
			<td style="width:180px;">04-100-812-E</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">fatty acid-free BSA</td>
			<td style="width:171px;">Roche</td>
			<td style="width:180px;">10,775,835,001</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">palmitate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">P0500</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Deoxy-D-glucose, 2-[1,2-3H (N)]</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">NET328A001MC</td>
			<td style="width:257px;">Specific Activity: 5-10Ci (185-370GBq)/mmol, 1mCi (37MBq</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Cytochalasin B</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">c2743</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PICO PRIAS VIAL 6ml</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">6000192</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">ultima gold MW CA&#160;</td>
			<td style="width:171px;">PerkinElmer</td>
			<td>6013159</td>
			<td style="width:257px;">scintillation liquid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">b&#234;ta counter&#160;</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">2900TR</td>
			<td style="width:257px;"></td>
		</tr>
	</tbody>
</table>

glucose uptake measurement and response to insulin stimulation in in vitro cultured human primary myotubes

骨骼肌是哺乳动物中最大的葡萄糖沉积物，在很大程度上有助于葡萄糖的体内平衡。评估肌肉细胞的胰岛素敏感性与所有研究专门用于探索肌肉葡萄糖代谢和表征代谢改变相关。在肌肉细胞中，葡萄糖转运蛋白4型（GLUT4）蛋白质响应于胰岛素转移到质膜，从而允许葡萄糖大量进入细胞。肌肉细胞通过增加葡萄糖摄取速率来响应胰岛素的能力是量化肌肉细胞对胰岛素敏感性的标准读数之一。人原代肌管是一种合适的体外模型，因为细胞维持供体表型的许多特征，包括胰岛素敏感性。这种体外模型也适用于可能影响胰岛素反应性的任何化合物的测试。分化肌管中葡萄糖摄取率的测量反映胰岛素敏感性。 在这种方法中，人体原代肌细胞在体外培养以获得分化的肌管，并且测量有和没有胰岛素刺激的葡萄糖摄取率。我们提供了一个详细的方案来量化使用放射性标记的[ 3 H] 2-脱氧-D-葡萄糖（[ 3 H] 2dG）的被动和活跃的葡萄糖转运率。提供计算方法来量化活性基础和胰岛素刺激率以及刺激折叠。

在第3天，成肌细胞达到汇合（ 图1A ）。在这个阶段的成肌细胞通常是单核的。培养基改变，第8天分化完成（ 图1B ）（方案第2节）。分化5天后，将肌管对准并且通常多核化。在葡萄糖摄取速率测量之前，将人原代肌管进行棕榈酸酯或仅BSA处理。将细胞与BSA（PALM）或BSA单独（BSA）中的棕榈酸酯0.5mM孵育48小时。进行胰岛素刺激?...

Watch this Scientific Journal Video about Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes at JoVE.com

体外培养的人原代肌管的葡萄糖摄取测量和对胰岛素刺激的反应|生物学 |视频

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

在这种方法中，人原代肌细胞在体外培养以获得分化的肌管，并测量葡萄糖摄取率。我们提供了一个详细的方案，使用放射性标记的[ 3 H] 2-脱氧-D-葡萄糖来量化基础和胰岛素刺激状态的速率。

葡萄糖摄取测量和对胰岛素刺激的反应<em&gt;体外</em&gt;培养的人类初级肌管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

glucose-uptake-measurement-response-to-insulin-stimulation-in-vitro

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Biology

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Cellular Processes

Metabolism

SCIENTISTS IN ACTION

19.7K 次观看.  University of Lyon. 在这种方法中，人原代肌细胞在体外培养以获得分化的肌管，并测量葡萄糖摄取率。我们提供了一个详细的方案，使用放射性标记的[ 3 H] 2-脱氧-D-葡萄糖来量化基础和胰岛素刺激状态的速率。 

视频: Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

科研

生物学

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

细胞器运输的培养<em&gt;果蝇</em&gt;细胞:S2细胞系和原代神经元。

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

细胞质DNA的检测途径的刺激<em&gt;在体外</em&gt;和<em&gt;体内</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

沉默<em&gt; BRCA2</em&gt;以确定新的BRCA2调控的生物学功能在人体细胞培养

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...