作者没有什么可透露的。

1. INS-1E 细胞: 维护和电镀
<ol><li>在加湿37˚C/5% CO2孵化器中维护 INS-1E 细胞, 并用 RPMI 1640 培养基补充 5% (v/v) 热灭活胎牛血清, 2 毫米 l-谷氨酰胺, 10 毫米 HEPES, 1 毫米丙酮酸钠, 100 U/毫升青霉素/链霉素溶液, 50 µM β巯基乙醇。培养细胞在10毫升完成 RPMI 1640 中等 (每板), 直到他们到达 80-90% 汇合, 当他们可以是 trypsinized 和传代或用于胰岛素分泌试验。</li>
	<li>
1 天:吸入介质, 用5毫升预热的 PBS 冲洗细胞一次。加入0.5 毫升胰蛋白酶 (0.025%) 稀释1:1 在0.5 毫升 PBS 胰蛋白酶处理细胞和孵育 3-4 分钟在37˚C。停用胰蛋白酶, 加入9毫升完整的培养基, 将细胞转移到15毫升离心管吹打。</li>
	<li>颗粒细胞通过离心和重新悬浮细胞颗粒在大约5毫升新鲜的媒介。</li>
	<li>取10µL 的重悬浮细胞, 并与10µL 台盼蓝生命染料混合, 检查细胞的活力。使用 hemocytometer 对10µL 中的活细胞和死体细胞进行计数。生存能力水平应高于90%。将新鲜培养基中的重悬浮细胞稀释至每毫升100万细胞。</li>
	<li>种子0.5 毫升 INS-1E 细胞每井在聚 l-赖氨酸预涂覆的24井板, 在密度50万细胞/好。</li>
	<li>
2 天:去除介质 18-24 小时后电镀和添加500µL/井新鲜 RPMI 1640 媒体。将细胞孵化为另外24小时, 使细胞从先前的传代步骤中完全恢复。这个额外的时间允许细胞在组织培养板上传播。</li>
</ol>2. 胰岛素分泌物测定 (3 天)
<ol><li>准备 KRB 缓冲器: 132.2 毫米氯化钠, 3.6 毫米氯化钾, 5 毫米 NaHCO3, 0.5 毫米 NaH2PO4, 0.5 毫米氯化镁2, 1.5 毫米 CaCl2, 0.001 克/毫升牛血清白蛋白 (BSA), pH 7.4。</li>
	<li>从细胞中吸取介质, 用预热过的 PBS 冲洗两次。</li>
	<li>对于葡萄糖饥饿步骤, 在37˚C/5% CO2中添加450µL/井 KRB (含 BSA), 无葡萄糖1小时。</li>
	<li>在葡萄糖饥饿的步骤, 准备稀释的药物 (s) 在 KRB 含有200毫米葡萄糖 (10x 浓度)。<ol>
<li>准备药物在10x 最后的集中在 KRB 补充与200毫米葡萄糖 (也10x 最后的化验葡萄糖集中)。如果药物库存 (加上10x 葡萄糖) 在亚砜, 确保亚砜的百分比保持一致的整个化验 (理想的最终百分比小于0.1% 亚砜)。</li>
		<li>对于多巴胺和 quinpirole 治疗, 使用最后的检测药物浓度范围为100µM 到 100 pM (从最高到最低浓度), 最后一点的剂量反应不含药物。对于溴隐亭, 使用最后的检测浓度范围为10µM 到下午10点, 最后一点的剂量反应是无药控制。</li>
	</ol>
</li>
	<li>葡萄糖饥饿后, 加入药物序列稀释的化验, 以产生剂量反应。</li>
	<li>添加50µL/井每系列稀释到相应的水井 (总化验量500µL)。</li>
	<li>对于葡萄糖刺激步骤, 孵化细胞与各自药物系列稀释 (在20毫米葡萄糖存在) 90 分钟在37˚C/5% CO2.包括一套控制井: (1) 在没有任何额外的药物和 (2) 不受药物或葡萄糖刺激的细胞 (提供基本分泌速率) 的情况下, 单独刺激20毫米葡萄糖。</li>
	<li>在刺激步骤之后, 小心地取出上清液 (直接使用或存储在4˚C)。 注: 此时可引入额外的5分钟温和离心步骤 (600 x g, 1 分钟), 以去除检测上清液中的剩余细胞。</li>
</ol>3. HTRF 测定胰岛素分泌物
<ol><li>稀释试验上清液1:10 在 KRB (不 BSA), 优选在清楚的96井板材。</li>
	<li>准备胰岛素标准曲线的 HTRF 胰岛素检测 (表 1)。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>标准库存解决方案 500 ng/毫升</td>
			<td>串行稀释</td>
			<td>工作 [胰岛素] ng/毫升</td>
		</tr>
<tr>
<td>性病7</td>
			<td>30µl 库存 + 140 µl KRB</td>
			<td>150</td>
		</tr>
<tr>
<td>性病6</td>
			<td>30µl 性病 7 + 45 µl KRB</td>
			<td>60</td>
		</tr>
<tr>
<td>性病5</td>
			<td>30µl 性病 6 + 45 µl KRB</td>
			<td>24</td>
		</tr>
<tr>
<td>性病4</td>
			<td>30µl 性病 5 + 45 µl KRB</td>
			<td>9。6</td>
		</tr>
<tr>
<td>性病3</td>
			<td>30µl 性病 4 + 45 µl KRB</td>
			<td>3.84</td>
		</tr>
<tr>
<td>性病2</td>
			<td>30µl 性病 3 + 45 µl KRB</td>
			<td>1.54</td>
		</tr>
<tr>
<td>性病1</td>
			<td>30µl 性病 2 + 45 µl KRB</td>
			<td>0.61</td>
		</tr>
<tr>
<td>性病0</td>
			<td>45µl KRB</td>
			<td>0</td>
		</tr>
<tr>
<td colspan="3">注: 性病股为500毫升</td>
		</tr>
</tbody></table>表1。串行稀释, 使胰岛素标准曲线。
<ol start="3"><li>将标准曲线样品和稀释后的测定上清液到 HTRF 板上。用 HTRF 测定分泌胰岛素的方法可以在96井或384井板中进行, 同时要记住化验量必须重新调整。使用10µL/井样品在96井白色半区域板材或5µL/井在384井白色低容量, 圆底板 (参见材料表)。</li>
	<li>在1:2 捐赠者 (cryptate)/受体 (XL-665) 比率中准备抗体组合, 检测缓冲区 (参见材料表)。 注: 有关 HTRF 化验的进一步具体细节可从制造商处获得。</li>
	<li>将抗体组合添加到30µL/井 (96 井板化验格式) 或15µL/井 (用于384井板化验格式) 的化验。</li>
	<li>把盘子封上, 室温下孵化。</li>
	<li>阅读板后2小时, 4 小时, 和/或夜间孵化与抗体使用的板读取器和适当的 HTRF 光学模块 (337 665 620 nm) (见材料表和制造商的说明)。将集成开始时间设置为60µs, 并在400µs 中集成. 使用200闪烁每井。 注: 这些参数是基于使用我们的特定读者。读数在620毫微米和665毫微米可能变化在不同的仪器之间。这是我们建议使用665/620 比率的原因之一。在计算这一比例时, 读者与读者之间的任何潜在差异都将被规范化, 并提供一致的价值, 而不管用来测量 HTRF 的仪器是什么。</li>
</ol>4. 数据分析和规范化
<ol><li>通过外推比例荧光读数 (665 nm/620 nm) 到二阶二次多项式曲线 (图 2) 计算出检测井的胰岛素浓度。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57531/57531fig2.jpg"> 图 2: 胰岛素标准曲线.采用已知浓度的人胰岛素储备, 生成胰岛素标准曲线。结果 HTRF 比率 (665 毫微米/620 毫微米) 被绘了反对胰岛素浓度。数据最适合于第二阶二次多项式曲线 (R2 = 0.99996)。这是一个具有代表性的标准曲线。误差线 = SEM.<a href="/files/ftp_upload/57531/57531fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>以推断的数据作为胰岛素分泌的 ng/毫升, 规范化 (胰岛素分泌反应增加配体浓度) 到% 最大胰岛素分泌物检测井的平均值 (20 毫米葡萄糖单独情况)。</li>
	<li>从单个实验中使用曲线拟合 (R2) 计算板内变化。在实验中, 从实验内的重复项中派生出单个 R2值, 允许计算曲线拟合的平均值的标准误差。</li>
	<li>为确定 interplate 变化, 请使用来自至少三个单独实验的数据来计算集体曲线 R2值的平均值的标准误差。</li>
</ol>

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我们感谢尼古拉-皮埃尔 (Cisbio 生物鉴定) 的有益建议和皮埃尔 Maechler (日内瓦大学) 慷慨提供 INS-1E 细胞。这项工作得到了国防部的资助 (PR141292 授予中福), 以及匹兹堡基金会 (中福) 的 Emmerling 基金。

这里描述的 HTRF 胰岛素检测提供了一个快速, 有效的系统来衡量胰岛素分泌从一个培养细胞的系统。在其最重要的优势之一, 该检测提供了低背景信号, 由于高信噪比。此外, 我们已确认 HTRF 信号在较长时间内稳定 (> 24 小时)7。然而, 由于胰岛素结合单克隆抗体在检测后快速达到束缚饱和度, 抗体结合的动力学可能会受到环境温度或胰岛素分泌水平的变化的影响。因此, 我们在多个?...

对能量代谢的调节由一种主要的合成代谢荷尔蒙--胰岛素--进行微调。胰岛素是由胰β细胞合成和释放, 以响应增加的胞外葡萄糖水平。释放的胰岛素触发葡萄糖的摄取胰岛素敏感组织1,2。生理上, 这与饭后葡萄糖浓度升高有关, 其次是胰岛素分泌调节葡萄糖摄取。在葡萄糖稳态紊乱导致代谢损害达到高潮的胰岛素抵抗, 最终在2型糖尿病的发病2,3,4。
虽然胰岛素分泌已被广泛研究, 但其调控机制仍不清楚。一个重要的调查领域是通过 beta 细胞5,6,7,8, 识别出新的胰岛素分泌调节剂。这些研究需要更好地了解葡萄糖刺激与胰岛素分泌的耦合关系。因此, 准确监测和量化葡萄糖刺激胰岛素分泌 (GSIS) 水平的能力是必不可少的。然而, 迄今为止, 只有有限数量的方法可以使用胰岛素分泌细胞系和/或胰岛来量化 GSIS。一种是放射免疫 (RIA), 它利用放射性同位素标记的胰岛素和抗体。这一方法的主要局限性包括放射性材料的处理和处置造成的安全问题。此外, 这种方法是劳动密集型的, 涉及多长时间的洗涤和孵化步骤。酶联免疫吸附试验 (ELISA) 是另一种昂贵的和劳动密集型的方法, 利用抗体进行胰岛素检测。抗体亲和性和胰岛素识别效率的变化是这种方法的限制因素, 会影响结果的重现性。ELISA 和 RIA 都不是为高通量实验设计的。AlphaScreen 是一种用于检测和测量胰岛素分泌水平的均质检测方法。AlphaScreen 技术的基础是将环境氧转化为激发氧的单态态, 这种状态可以与化学发光物种发生反应, 从而产生化学发光。由于该检测是同源的, 许多与 RIA 和 ELISA 相关的洗涤步骤被消除。然而, 由于反应的性质, 信号的不稳定性是一个限制因素, 可能会影响检测的读数。(Heyduk 和同事9开发的 TR 钳, 是基于两个单独的抗体对胰岛素分子的不同表位进行绑定的另一种均匀的胰岛素测量方法。抗体每个化学上链接到双链 dna 与短互补单链 dna 悬。抗体与胰岛素的结合使它们聚集在一起, 导致双链 DNA 双工。每种抗体也与各自的捐献者或受体荧光相关联, DNA 双工的联想汇集这些显影产生 Förster 共振能量转移 (烦恼)。然而, 一个潜在的限制是 TR 钳, 取决于烦恼本身。在焦虑反应过程中, 无法迅速消散背景荧光, 可能导致实验中的背景荧光和低信噪比的水平较高。因此, 对于以高通量方式量化 GSIS 的可靠、稳健和经济高效的检测, 仍然需要存在。
生物物理学的最新进展最终导致了一种均匀时间分辨荧光能量共振转移 (HTRF) 的研究。具体来说, 虽然分析中的能量转移可以被描述为基于焦虑的, 更准确地说, HTRF 依赖于发光能量共振转移 (LRET)10 , 这是能量在供体和受体之间的非辐射传递。物种11,12,13。这一区别是重要的, 因为荧光或淬火基于焦虑交互的时间与 LRET 有很大的不同, 尽管同样类型的浇口可以用于烦恼和 LRET。此外, 稀土稀土 cryptate 化合物, 如铕或铽 cryptate 在 HTRF 中的使用产生长荧光半衰期12,14。这提供了在捐助者励磁与接受方 (即,时间分辨化验) 之间的排放量之间引入时间延迟 (µsec) 的独特优势。这个时间延迟允许在测量受体发射荧光之前有足够的时间让背景荧光消散。因此, 读数是没有非特定的荧光, 因此, 高信噪比达到 (图 1)。此外, HTRF 的均一性消除了清洗未绑定物种的洗涤步骤的需要, 使得该检测比 ELISA 法或基于 RIA 的方法要快得多。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57531/57531fig1.jpg"> 图 1: HTRF 胰岛素检测机制的示意图.两个独立产生的单克隆抗体专门识别和绑定到胰岛素在不同的地点。这些抗体是共轭的铕 cryptate 捐赠者或 XL665 受体。捐赠者的激发在337毫微米导致放射在620毫微米。由此产生的能量转移导致 XL665 以更长的波长发出, 665 nm。<a href="/files/ftp_upload/57531/57531fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在这里, 我们提供了一个详细的协议, 以使用基于 HTRF 的方法来确定 GSIS 的水平, 从 INS-1E 细胞, 一个建立良好的胰岛素分泌β细胞衍生的大鼠胰岛素瘤细胞系15。此外, 该检测可用于鉴定胰岛素分泌分子调控剂的药理学特征。我们应用这种 HTRF 的胰岛素检测, 以检查多巴胺 D2样受体调节 GSIS。越来越多的研究表明, 神经递质多巴胺是 GSIS8,16,17,18,19,20的重要调节器,21,22. 多巴胺以阴性分泌/分泌方式影响 GSIS, 通过对多巴胺 D2样受体 (d2, D3, d4受体) 的反应, 在β胰腺细胞的表面表达8,16,19. 利用这一方法, 我们确认多巴胺作为 GSIS 的负调节因子的作用, 并证明多巴胺 D2样受体激动剂溴隐亭和 quinpirole 也能减少 GSIS。

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well white half area plate</td>
			<td style="width:104px;">Greiner Bio-One</td>
			<td style="width:139px;">82050-042</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">384-well white low-volume, round-bottom plate</td>
			<td style="width:104px;">Corning</td>
			<td>15100157</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin High Range Kit</td>
			<td style="width:104px;">Cisbio Bioassays</td>
			<td style="width:139px;">62IN1PEH&#160;</td>
			<td>10,000 test HTRF-based insulin assay kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PHERAstar FSX plate reader</td>
			<td style="width:104px;">BMG Labtech</td>
			<td>FSX</td>
			<td>Plate reader adapted for HTRF-based assay readings</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plate sealers</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:139px;">DY992</td>
			<td>To seal plate while antibodies incubate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hemocytometer</td>
			<td style="width:104px;">Hausser Scientific</td>
			<td style="width:139px;">3100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI Medium 1640 (1x)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11875-093</td>
			<td>[+] L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 0.05%&#160;</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">25-052-CI</td>
			<td>0.53 mM EDTA, (-) sodium bicarbonate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DPBS (Dulbecco&#39;s phosphate buffered saline)</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">21-031-CV</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">35-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">156-30-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11360070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin/Streptomycin solution 100x</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">30-002 CT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">M1348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dopamine hydrochloride</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">8502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromocriptine mesylate</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(-)-Quinpirole hydrochloride</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">1061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:104px;">Calbiochem</td>
			<td style="width:139px;">12659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly-L-Lysine</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">P4832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">276855</td>
			<td></td>
		</tr>
	</tbody>
</table>

homogeneous time-resolved f&#246;rster resonance energy transfer-based assay for detection of insulin secretion

胰岛素分泌的检测对于阐明调控分泌物的机制以及代谢研究至关重要。虽然许多胰岛素检测已经存在数十年, 最近出现的均匀时间分辨 Förster 共振能量转移 (HTRF) 技术已经大大简化了这些测量。这是一种快速、经济高效、重现性强的光学检测方法, 依赖于具有长持久辐射的明亮显影的抗体, 这有助于时间分辨 Förster 共振能量转移。此外, HTRF 胰岛素检测也适合于高通量筛查技术的发展。在这里, 我们使用 HTRF 检测胰岛素分泌的 INS-1E 细胞, 一个大鼠胰岛素瘤衍生细胞系。这使我们能够估计胰岛素的基本水平及其对葡萄糖刺激反应的变化。此外, 我们使用这个胰岛素检测系统来确认多巴胺作为葡萄糖刺激胰岛素分泌的负调节因子 (GSIS) 的作用。类似地, 其他多巴胺 D2样受体激动剂, quinpirole 和溴隐亭, 以浓度依赖性的方式减少 GSIS。我们的研究结果突出了 HTRF 胰岛素检测格式在确定 GSIS 中多种药物的作用及其药理学特征方面的效用。

我们通过使用纯化的人胰岛素标准的预定义浓度 (图 2) 生成胰岛素标准曲线来验证我们的胰岛素 HTRF 检测。标准曲线的生成允许我们推断比例荧光读数, 从而确定分泌的胰岛素水平, 以应对药物治疗 (图 2)。曲线拟合的板内变化最小 (R2 = 0.99996 5.0 x 10-5) interplate 变化 (R2 = 0.947 @ 5.5 x 10-5</sup...

Watch this Scientific Journal Video about Homogeneous Time-resolved FÃƒÂ¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion at JoVE.com

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

在这里, 我们提出的均匀时间解决烦恼 (HTRF) 作为一种有效的方法来快速检测胰岛素分泌的细胞。

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

homogeneous-time-resolved-frster-resonance-energy-transfer-based

Research

JoVE Journal

Medicine

9.1K 次观看.  University of Pittsburgh. 在这里, 我们提出的均匀时间解决烦恼 (HTRF) 作为一种有效的方法来快速检测胰岛素分泌的细胞。

视频: Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

科研

医学

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

提高在社区医院静脉注射胰岛素政府

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

术中的微妙的子宫内膜异位症的检测:A新型范式为相关联的与的的腹膜的完整性之亏损盆腔疼痛的的的的检测和治疗的

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

共培养影响胰腺β细胞的胰岛素分泌细胞外蛋白质相互作用分析

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

7号特斯拉的心脏磁共振成像

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

危重病人全血内毒素血症检测的内毒素活性测定

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

利用隔离的完美大鼠小肠进行肠道激素分泌的机制

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...