作者要感谢 Tessem 和汉考克实验室的成员们进行了辅助和科学的讨论。作者感谢安德鲁尼尔逊博士 (弗吉尼亚理工学院) 提供可可衍生的儿茶单体分数。这项研究得到了来自糖尿病行动研究和教育基金会对 JST 的资助。

1. 细胞培养
<ol><li>培养 INS-1 获得832/13 β细胞在 RPMI 1640 补充与10% 胎牛血清 (FBS), 1% 青霉素-链霉素, 10 毫米 HEPES, 2 毫米谷氨酰胺, 1 毫米钠丙酮酸盐, 和0.05 毫米 2-基9,10,11 ,12,13。</li>
	<li>用2毫升0.25% 的胰蛋白酶从 T75 烧瓶中取出832/13 细胞, 在37° c 下孵育10分钟, 加入8毫升的完整 RPMI 1640 介质。</li>
	<li>计算细胞使用例和稀释100µL 的细胞体积从步骤1.2 在900µL 的 PBS。</li>
	<li>在6井盘中, 在密度为 2 x 106细胞/mL 的情况下, 将832/13 细胞板。</li>
	<li>在开始呼吸实验之前, 在37° c 和 5% CO2的湿恒温箱中培养细胞为 48 h。</li>
</ol>2. 高分辨率呼吸细胞的制备
<ol><li>
用可可儿茶单体处理832/13 细胞。
	<ol>
<li>在37° c 和 5% CO2的湿恒温箱中电镀后培养832/13 细胞24小时。</li>
		<li>改变媒体24小时后电镀, 并处理832/13 细胞与车辆控制 (3 口井) 或100纳米可可单体 (3 口井), 其次是文化的一个额外的 24 h (48 h 共计) 在湿化孵化器在37° c 和 5% CO2。</li>
		<li>在1x 低葡萄糖分泌物检测缓冲液中洗涤832/13 细胞, 在1x 低血糖的5分钟内孵育832/13 细胞, 每小时改变1x 低血糖浓度。<ol>
<li>用33.32 克氯化钠, 1.73 克氯化钾, 0.82 克2PO4, 0.7 克 MgSO4 , 并将 H2O 添加到最终的500毫升。无菌过滤器的最终解决方案。</li>
			<li>用10毫升10x 的低血糖的 1x, 100 µL 45% 葡萄糖, 2 毫升的1米 HEPES, 1 毫升的 0.25M CaCl2, 0.57 毫升的 35% BSA 溶液, 0.21 g 的 NaHCO3 , 并添加 H2O 到最后的体积100毫升。无菌过滤器的最终解决方案。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
用姜黄素治疗832/13 细胞。
	<ol>
<li>在37° c 和 5% CO2的湿恒温箱中电镀后培养832/13 细胞48小时。</li>
		<li>在1x 低血糖的5分钟内冲洗832/13 细胞, 在1x 的低血糖浓度下孵育832/13 细胞, 每小时更换1x 低血糖。</li>
		<li>在1x 低血糖的 3 h 孵化后 2.5 h, 吸气缓冲和孵育细胞在1x 低血糖审计与车辆控制或40µM 姜黄素30分钟。继孵育后, 吸气缓冲液。</li>
	</ol>
</li>
	<li>
高分辨率呼吸的胰蛋白酶捕获细胞
	<ol>
<li>在1x 低血糖的 3 h 孵化后, 从板上取出细胞, 每井250µL 0.25% 胰蛋白酶。</li>
		<li>在15毫升锥形管中, 将胰酶中的细胞与汽车控制或复利的4毫升的3口井结合起来。</li>
		<li>使用例的计数细胞和稀释100µL 的细胞体积从步骤2.3.2 在900µL PBS。</li>
		<li>稀释1x 低血糖局的适当数量的细胞, 使每个腔室的适当浓度为3毫升。数据显示 1 x 106单元/mL 的浓度是最有效的。</li>
	</ol>
</li>
	<li>
高分辨率呼吸的机械离解收获细胞
	<ol>
<li>在1x 低血糖的 3 h 孵化后, 增加1毫升的1x 低血糖, 每井, 轻轻地吹离板的细胞与机械离解移与1000µL 吸管尖端。</li>
		<li>将3口井中的细胞与车辆控制或复利结合在一起, 在15毫升的锥形管中进行呼吸, 共3毫升。</li>
	</ol>
</li>
</ol>3. 高分辨率呼吸
<ol><li>高分辨率呼吸的制备。
	<ol>
<li>打开高分辨率呼吸并通过启动呼吸分析程序连接到桌面。</li>
		<li>从两个腔中抽出70% 乙醇。在70% 乙醇中浸泡这些腔至少45分钟。</li>
		<li>用70% 乙醇洗两次腔, 每步吸气。</li>
		<li>洗涤室三次与 ddH2O, 吸气后, 每一步。</li>
		<li>冲洗活塞在 ddH2O 中的腔室。这将清除活塞和钛港残留的乙醇。</li>
	</ol></li>
	<li>极谱氧传感器的校准。
	<ol>
<li>在每个 oxygraph 室中加入2.4 毫升的1x 低葡萄糖浓度的缓冲液, 在 750 rpm 和37° c 的情况下, 用磁搅拌棒连续搅动缓冲液, 在2.0 秒内通过推 F7 按钮并打开标签为 "系统" 的选项卡来记录数据间隔。推活塞所有的方式, 然后收回到扳手曝气设置。让机器平衡至少1小时, 直到获得稳定的氧气流量。</li>
		<li>在实验期间, 将机器设置在37° c。将极化电压设置为 800 mV, 通过推 F7 按钮, 并打开标签为 "氧气, O2" 的标签, 增益为2。平衡至少30分钟的氧浓度, 而氧浓度的变化是稳定的 (少于 2 pmol/毫升)。</li>
		<li>在氧浓度稳定后, 选择氧浓度变化稳定的区域, 建立氧浓度变化的背景测量。<ol>
<li>通过推送 shift 键选择一个区域, 单击鼠标左键并在选定区域中拖动鼠标。单击与所选区域关联的字母, 并更改为每个跟踪的 "R1", 对应于两个分庭。</li>
			<li>双击屏幕左下方和右下角的 "O2校准" 框, 单击 "选择标记" 按钮以将 "空气校准" 作为 R1, 然后为两个腔室选择 "校准并复制到剪贴板"。</li>
		</ol>
</li>
	</ol></li>
	<li>2.1.5 或2.2.5 步骤制备的832/13 种β细胞的呼吸评价。
	<ol>
<li>在每个腔室中加载2.4 毫升的样品 (一个室与控制车辆处理的细胞, 一个室与复合处理的细胞) 在1x 低血糖审计。如果使用机械离解法 (在-20 ° c 下冻结测量), 将0.5 毫升的细胞样本用于蛋白质定量测定 (二辛可宁酸)。<ol>
<li>推动柱塞的所有方式, 并吸取残余量。在所有后续步骤中, 在 750 rpm 和37° c 的整个实验中连续搅拌细胞。在加载示例时, 单击 F4 并将其标记为 "单元格" 以进行标记。</li>
			<li>测量样品30分钟。在信号稳定后, 选择一个区域的氧浓度变化对应的低葡萄糖条件 (2.5 毫米葡萄糖) 描述的3.2.3。</li>
		</ol>
</li>
		<li>在信号稳定达到后, 添加12.5 µL 45% 无菌葡萄糖溶液 (16.7 毫米最终浓度) 进入每个房间通过钛装货港使用注射器。在添加治疗时, 用3.3.1 描述的 "葡萄糖" 作标记。让信号稳定并记录细胞呼吸, 直到达到稳定的氧气流量。选择这个区域的氧气浓度的变化, 如3.2.3 所述。这是 16.7 mM 葡萄糖读数, 并与刺激条件7对应。</li>
		<li>在信号稳定以后被到达增加1µL 5mM 寡 A (2.5 µM 最后的集中) 入每个房间通过装货口岸。在添加治疗时, 用3.3.1 描述的 "OligoA" 作标记。让信号稳定并记录细胞呼吸, 直到达到稳定的氧气流量。选择曲线的这一区域, 如3.2.3 中所述。寡抑制 ATP 合成酶, 因此唯一的氧气通量发生是通过电子泄漏和不氧化磷酸化7。</li>
		<li>在信号稳定被到达之后增加1毫米 FCCP 在1µL 增量直到最大呼吸率建立。这代表最大的不耦合呼吸作用。3-4 µL FCCP 是足够的 (1.5-2.0 µM 最终浓度), 以诱导最大的非耦合呼吸 INS-1 832/13 细胞。在添加治疗时, 将标记标记为 "FCCP", 如3.3.1 所述。让信号稳定并记录细胞呼吸, 直到达到稳定的氧气流量。选择曲线的这一区域, 如3.2.3 中所述。FCCP 是一个解耦代理。这使我们可以测量不耦合呼吸7。</li>
		<li>在信号稳定以后被到达增加1µL 5 毫米 Antimycin A (2.5 µM 最后的集中) 入每个房间通过装货口岸。在添加治疗时, 将标记标记为 "AntiA", 如3.3.1 所述。让信号稳定并记录细胞呼吸, 直到达到稳定的氧气流量。选择曲线的这一区域, 如3.2.3 中所述。 注: Antimycin A 结合细胞色素 C 还原酶, 抑制泛氧化, 并阻断所有呼吸。这使我们能够完全停止呼吸7。</li>
	</ol></li>
	<li>测量832/13 β细胞的蛋白质浓度和呼吸正常化。
	<ol>
<li>使用0.5 毫升样本保留在装货, 测量蛋白质样本使用的评估方法13。</li>
		<li>按照制造商的指示, 在不稀释的情况下, 每三份样品运行一次。</li>
	</ol></li>
	<li>832/13 β细胞呼吸计算从 trypsinized 细胞。<ol>
<li>通过推呼吸分析程序的 F3 按钮, 在检测中输入每毫升使用的细胞数。将单位改为细胞/毫升, 进入细胞浓度, 并改变介质到审计。</li>
		<li>选择背景读数以使数据正常化, 方法是选择并输入 O2校准表单, 如3.2.3 中所述。</li>
		<li>从2.5 毫米葡萄糖, 16.7 毫米葡萄糖, 寡 a, FCCP 和 Antimycin a 的读数进行选择, 如3.3.1 所述。</li>
		<li>在呼吸分析程序中, 在进入蛋白质浓度后, 为每个腔室输出读数, 并在呼吸测量期间选择适当的平均值。这是通过单击 F2, 然后推动 "复制到剪贴板功能" 完成的。这将导出要在其他分析程序中使用的数据。使用 "O2斜率为负值" 进行计算。</li>
		<li>为3-5 独立运行的车辆处理控制和天然化合物处理细胞的数据, 以确定对 INS-1 832/13 β细胞的完整细胞呼吸的影响。</li>
	</ol></li>
	<li>832/13 β细胞呼吸计算从机械上离解的细胞。<ol>
<li>通过推呼吸分析程序的 F3 按钮, 从该方法中输入蛋白质计算。将单位改为 mg, 进入其浓度, 并将培养基转变为审计组。</li>
		<li>选择背景读数以使数据正常化, 方法是选择并输入 O2校准表单, 如3.2.3 中所述。</li>
		<li>从2.5 毫米葡萄糖, 16.7 毫米葡萄糖, 寡 a, FCCP 和 Antimycin a 的读数进行选择, 如3.3.1 所述。</li>
		<li>在呼吸分析程序中, 在进入蛋白质浓度后, 为每个腔室输出读数, 并在呼吸测量期间选择适当的平均值。这是通过单击 F2, 然后推动 "复制到剪贴板功能" 完成的。这将导出要在其他分析程序中使用的数据。使用 "O2斜率为负值" 进行计算。</li>
		<li>为3-5 独立运行的车辆处理控制和天然化合物处理细胞的数据, 以确定对 INS-1 832/13 β细胞的完整细胞呼吸的影响。</li>
	</ol></li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resveratrol+and+curcumin+enhance+pancreatic+beta-cell+function+by+inhibiting+phosphodiesterase+activity.">Resveratrol and curcumin enhance pancreatic beta-cell function by inhibiting phosphodiesterase activity.</a> J Endocrinol. 223 (2), 107-117 (2014).</li><li>Ma, Z., 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=Diabetes+reduces+beta-cell+mitochondria+and+induces+distinct+morphological+abnormalities,+which+are+reproducible+by+high+glucose+in+vitro+with+attendant+dysfunction.">Diabetes reduces beta-cell mitochondria and induces distinct morphological abnormalities, which are reproducible by high glucose in vitro with attendant dysfunction.</a> Islets. 4 (3), 233-242 (2012).</li><li>Chavanelle, V., 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=Effects+of+high-intensity+interval+training+and+moderate-intensity+continuous+training+on+glycaemic+control+and+skeletal+muscle+mitochondrial+function+in+db/db+mice.">Effects of high-intensity interval training and moderate-intensity continuous training on glycaemic control and skeletal muscle mitochondrial function in db/db mice.</a> Sci Rep. 7 (1), 204 (2017).</li><li>Jelenik, T., 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=Mechanisms+of+Insulin+Resistance+in+Primary+and+Secondary+Nonalcoholic+Fatty+Liver.">Mechanisms of Insulin Resistance in Primary and Secondary Nonalcoholic Fatty Liver.</a> Diabetes. 66 (8), 2241-2253 (2017).</li><li>Spacek, T., 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=Glucose-stimulated+insulin+secretion+of+insulinoma+INS-1E+cells+is+associated+with+elevation+of+both+respiration+and+mitochondrial+membrane+potential.">Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential.</a> Int J Biochem Cell Biol. 40 (8), 1522-1535 (2008).</li><li>Jezek, J., Dlaskova, A., Zelenka, J., Jaburek, M., Jezek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2O2-Activated+Mitochondrial+Phospholipase+iPLAgamma+Prevents+Lipotoxic+Oxidative+Stress+in+Synergy+with+UCP2,+Amplifies+Signaling+via+G-Protein-Coupled+Receptor+GPR40,+and+Regulates+Insulin+Secretion+in+Pancreatic+beta-Cells.">H2O2-Activated Mitochondrial Phospholipase iPLAgamma Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic beta-Cells.</a> Antioxid Redox Signal. 23 (12), 958-972 (2015).</li><li>Johnson, J. D., 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=Suppressed+insulin+signaling+and+increased+apoptosis+in+CD38-null+islets.">Suppressed insulin signaling and increased apoptosis in CD38-null islets.</a> Diabetes. 55 (10), 2737-2746 (2006).</li></ol>

作者没有什么可透露的。

本议定书的目的是使用高分辨率呼吸测量完整的胰β细胞的呼吸速率。这种方法允许测量β细胞的反应, 以增加葡萄糖水平。该协议还允许对各种化合物进行预处理, 如本协议所示, 与自然发生的单体儿茶或姜黄素4,5。与其他各种化合物的治疗可以用于本议定书, 与预处理 (如此处所示), 或通过对基本协议的最小修改在呼吸室内治疗。
可以通过多种方法修改此方法。可使用其他β细胞系;然而, 需要通过实验来确定细胞数和洗净时间。也可以使用主要的啮齿动物和人类胰岛;然而, 细胞数量或胰岛当量也需要通过实验来确定。至少有一项研究使用高分辨率呼吸15测量了胰岛的耗氧量。这项研究要求每反应400胰岛, 但没有测量葡萄糖诱发的呼吸变化。鉴于主要组织经常使用高分辨率 respirometers16,17, 完整的小岛可以在实验故障诊断后使用。然而, 鉴于该试验所需的胰岛数量众多, 其他测量氧耗的方法可能更适合于原发性胰岛呼吸的测量。
有几个关键步骤与提出的协议。首先, 有足够数量的细胞是至关重要的。我们的研究表明, 832/13 β细胞的浓度 1 x 106细胞/毫升是足够的葡萄糖刺激呼吸测量。测量使用 0.5 x 106细胞/毫升显示低葡萄糖刺激呼吸和低信噪比。此外, 测量使用 2 x 106细胞/毫升导致高样本的样本变异性, 并迅速细胞使用的氧气, 需要室重新氧, 并经常导致快速细胞死亡。为了对这一关键步骤进行故障诊断, 应在给定的细胞线上实验确定适当数量的细胞。
第二个关键步骤是准备β细胞, 以应对高血糖水平。RPMI 1640 培养基具有较高的葡萄糖水平。细胞必须从高糖转移到低葡萄糖文化。一个 3 h 孵化在1x 低葡萄糖审计是足够的β细胞有一个显着的反应, 增加了 ~ 16.7 毫米葡萄糖。洗涤期少于3小时, 结果在变量的反应, 以增加葡萄糖。解决葡萄糖反应问题可以通过增加的时间长度的审计。
第三个关键步骤是为测量单元准备的方法。我们的数据表明, 虽然胰蛋白酶和机械离解都可以使用, 胰蛋白酶释放细胞的结果更一致的读数和较少样本-样品的变异性。这两种方法观察到的趋势都得到了维持, 然而数据的强度与使用胰蛋白酶制备的细胞更大。虽然其他方法, 如细胞刮, 可以使用我们的数据表明, 使用胰蛋白酶将导致最可重现的结果, 以较少样品样本的变异性18,19。
此方法的局限性包括以下几方面。首先, 只有两个治疗室, 这不是一个高通量筛选方法。每次运行两个样本之间需要3-4 小时, 包括准备机器的时间。其次, 由于每室所需的样本量为2.2 毫升 (或 2.2 x 106细胞), 因此化验需要相对较大的细胞浓度。由于化验所需的样品量, 除非有大量供应, 否则对主胰岛的此工具和协议的使用是有限的。以前的研究, 使用胰岛呼吸在这项技术使用〜400小岛/室15。这将需要, 大致上, 小岛从两只老鼠为每个房间20。第三, 由于化细胞没有被使用, 没有细胞渗透性的化合物来确定对单个电子链组件的处理效果的能力是不可用的。
本议定书在现有方法方面有许多重要的优点。首先, 该协议允许对各种膜渗透化合物进行逐步滴定。这使得准确确定必要的浓度。其次, 使用完整的β细胞可以让我们查询对胞浆和线粒体葡萄糖代谢的影响7。这使我们可以观察表型酵通路的变化, 这是一种在使用化细胞时丢失的观察。第三, 完整的细胞允许我们使用对高血糖的反应作为β细胞功能的量度, 这是失去了化变种的这种化验。最后, 该协议允许添加各种和多种化合物来查询化合物对β细胞呼吸的影响, 这是一种不存在于其他测量呼吸的工具的奢侈品。
此方法的未来应用包括能够测量诸如 ATP、钙和 H2O2级别的参数, 同时进行呼吸测量。这种方法还允许在其他燃料源 (如脂肪酸或酮) 存在的情况下测量呼吸。最后, 通过一些修改, 测量分泌的胰岛素水平伴随呼吸测量也可能是可能的。

胰β细胞的主要目的是维持系统血糖通过葡萄糖刺激的胰岛素分泌。β细胞感觉循环葡萄糖的生理变化主要归结于低亲和力, 高容量葡萄糖转运器 GLUT2 (葡萄糖运输者 2, Km 16.7 毫米)1。随着循环葡萄糖水平的升高, 这种高容量低亲和力转运体促进了β细胞内葡萄糖的比例增加。葡萄糖通过糖酵解、TCA 循环 (三酸循环) 和线粒体呼吸导致细胞 ATP (三磷酸腺苷) 水平的升高而代谢。atp 浓度升高会阻止 atp 敏感的 K+通道, 从而导致膜去极化。膜去极化导致电压门控 Ca 的开放2 +通道和随后释放囊泡胰岛素颗粒2。β细胞功能障碍是2型糖尿病 (T2D) 的标志, 其结果是减少和控制不良的胰岛素分泌和最终的β细胞死亡3。维持或改善β细胞功能的机制可用于治疗 T2D。
研究已经证明了自然发生的植物基化合物对胰腺β细胞4的有益作用。这些化合物可能通过增加β细胞增殖、存活或葡萄糖刺激的胰岛素分泌而产生作用。例如, 最近的研究表明, 单体儿茶通过增加线粒体呼吸和增加细胞 ATP 水平来增强葡萄糖刺激的胰岛素分泌5。因此, 了解这些化合物如何能增加功能性β细胞质量, 是很重要的利用这些化合物作为潜在的疗法。
细胞呼吸可以通过一些工具来测量。使用高分辨率呼吸允许滴定化学调节剂到化或完整的细胞数量6。该工具允许在不同浓度下添加各种化合物, 从而提供广泛的信息。
鉴于葡萄糖代谢和β细胞功能之间的密切联系, 细胞呼吸的测量是至关重要的。细胞呼吸的测量可以使用化或完整的β细胞进行, 每一个都有自己的优缺点7,8。虽然β细胞的性允许人们测量电子传输链的不同方面, 但它不考虑在β细胞、葡萄糖摄取和新陈代谢中诱导呼吸的机制。因此, 使用 unpermeabilized β细胞呼吸是一种非常有用的技术来确定β细胞对各种葡萄糖水平的反应, 使用氧气消耗量作为读数。
该技术的目的是测量完整的 INS-1 832/13 β细胞的耗氧量。这项技术使我们能够确定β细胞对 unstimulatory 葡萄糖 (2.5 mm 葡萄糖) 的反应以及刺激葡萄糖条件 (16.7 毫米葡萄糖)。虽然 unpermeabilized 细胞不允许我们单独测试电子传输链的复杂 I、II 或 III, 但该技术允许测量复杂的 IV 抑制 (寡 A), 不耦合呼吸 (FCCP-羰基氰化物-4-三氟甲氧基腙), 完全抑制呼吸 (Antimycin A)。这项研究证明了测量呼吸在完整的 unpermeabilized 胰腺β细胞的功效, 以及两种自然发生的化合物, 单体儿茶和姜黄素对β细胞呼吸的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>O2k Core: Oxygraph-2k</td>
			<td>Oroboros Instruments</td>
			<td>10000-02</td>
			<td>Instrument for high-resolution respirometry</td>
		</tr>
		<tr>
			<td>DatLab 6 Program</td>
			<td>Oroboros Instruments</td>
			<td>27141-01</td>
			<td>Computer program for analysing high-reolution respirometry</td>
		</tr>
		<tr>
			<td>INS-1 832/13 cell line</td>
			<td>Duke University Medical Center</td>
			<td>NONE</td>
			<td>Beta cell line, gift from Dr. Christopher Newgard</td>
		</tr>
		<tr>
			<td>Curcumin</td>
			<td>Sigma</td>
			<td>C7727</td>
			<td>Pre-treatment of beta cells</td>
		</tr>
		<tr>
			<td>Cocoa epicatechin monomer</td>
			<td>Virginia Polytechnic Institute and State University</td>
			<td>NONE</td>
			<td>Pre-treatment of beta cells, gift from Dr. Andrew Neilson</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td>832/13 beta cell media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td>832/13 beta cell media component</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P4458</td>
			<td>832/13 beta cell media component</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3662</td>
			<td>832/13 beta cell media component/ 1x SAB Buffer</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Caisson</td>
			<td>GLL01</td>
			<td>832/13 beta cell media component</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td>832/13 beta cell media component</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td>832/13 beta cell media component</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271</td>
			<td>Component of 10x SAB buffer</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>Component of 10x SAB buffer</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>Component of 10x SAB buffer</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td>Component of 10x SAB buffer</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose Solution</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td>Component of 1x SAB buffer, chemical for respiration assay</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C5670</td>
			<td>Component of 1x SAB buffer</td>
		</tr>
		<tr>
			<td>35% BSA Solution</td>
			<td>Sigma</td>
			<td>A7979</td>
			<td>Component of 1x SAB buffer</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>Component of 1x SAB buffer</td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>Sigma</td>
			<td>459844</td>
			<td>Washing Oxygraph O2k Chambers</td>
		</tr>
		<tr>
			<td>Oligomycin A</td>
			<td>Sigma</td>
			<td>O4876</td>
			<td>Chemicals for respiration assay</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma</td>
			<td>C2920</td>
			<td>Chemicals for respiration assay</td>
		</tr>
		<tr>
			<td>Anitmycin A</td>
			<td>Sigma</td>
			<td>A8674</td>
			<td>Chemicals for respiration assay</td>
		</tr>
		<tr>
			<td>BCA Protein Kit</td>
			<td>Thermo Fisher</td>
			<td>23225</td>
			<td>For determining protein concentration</td>
		</tr>
	</tbody>
</table>

high-resolution respirometry to measure mitochondrial function of intact beta cells in the presence of natural compounds

高分辨率呼吸允许测量分离的线粒体、细胞和组织的耗氧量。β细胞通过控制血糖水平, 通过胰岛素分泌来调节血糖浓度, 从而在体内发挥关键作用。胰岛素分泌受葡萄糖代谢和线粒体呼吸的控制。因此, 测量完整的β细胞呼吸是必不可少的, 以提高β细胞功能作为治疗糖尿病。使用完整的 832/13 INS-1 衍生的β细胞, 我们可以测量增加葡萄糖浓度对细胞呼吸的影响。这个协议允许我们测量β细胞呼吸在存在或没有各种各样的化合物, 允许一个确定特定化合物的作用在原封细胞呼吸。在这里, 我们展示了两种自然发生的化合物, 单体儿茶和姜黄素, 对β细胞呼吸在低 (2.5 毫米) 或高糖 (16.7 毫米) 条件下的作用。这种技术可以用来确定不同的化合物对完整的β细胞呼吸在不同的葡萄糖浓度存在的影响。

INS-1 832/13 β细胞的准备和收获的协议中所描述的, 将显示在氧气消耗的基础上的各种化学干预 (图 1A)。当葡萄糖浓度增加到16.7 毫米葡萄糖 (图 1B) 时, 将观察到呼吸的增加。当完整的细胞被寡处理后, 呼吸会减少。这种呼吸称为渗漏, 它被定义为基底, nonphosphorylating 呼吸状态。当β细胞被解偶联剂 FCCP, 即 ETS 呼吸或电子传输系统呼吸时, 将达到最大呼吸。最后, 呼吸将降到零, 当治疗与 Antimycin A, 标记为火箭 (残余氧气消耗量) 提到非呼吸的细胞氧气消耗量。
在细胞数量上的差异将会显著改变从该技术获得的数据的质量。葡萄糖诱导呼吸测量在 0.5, 1 和 2 x 106细胞/毫升 (图 2A-2C)。利用细胞计数和蛋白质检测, 计算出与三测试细胞浓度对应的蛋白质浓度 (图 2D)。使用 0.5 x 106细胞/毫升的测量未能证明葡萄糖刺激的呼吸 (图 2A), 而且只有 ETS 相关的增加呼吸被证明是显著的。这表明, 低的细胞数导致低信噪比, 混淆的结果。同样, 2 x 106单元/mL 的测量也导致显著的可变性, 这表明只有在 ETS 测量 (图 2C) 中才达到统计意义。这大概是由于需要在分析过程中多次重新氧, 这就导致了从样品到样品的更大变化。这些数据表明, 浓度 1 x 106细胞/毫升, 导致显著的葡萄糖刺激呼吸, 泄漏和 ETS 测量, 这是必要的呼吸分析 (图 2B和 2E)。
本协议提出了两种去除呼吸细胞的方法。当测量在37° c 完成时, 使用胰蛋白酶去除细胞是有效的。在缺乏或存在姜黄素 (图 3A) 的情况下进行的呼吸测量显示了葡萄糖刺激的呼吸、渗漏和 ETS 呼吸的维持。姜黄素治疗的细胞与未处理细胞的比较表明这些呼吸参数中没有任何变化 (图 3B-C)。在没有或存在可可儿茶单体 (图 3D) 的情况下进行的呼吸测量也表明了葡萄糖刺激的呼吸、渗漏和 ETS 呼吸的维持。可可儿茶单体处理过的细胞与未经治疗的细胞的比较表明, 在2.5 毫米和16.7 毫米葡萄糖, 以及在泄漏和 ETS 呼吸 (图 3E-f) 的呼吸增加。这些数据表明, 虽然姜黄素不提高832/13 β细胞呼吸, 可可儿茶单体确实 (图 3G)。姜黄素通过抑制磷酸二酯酶的活性而显示出增强胰岛素分泌的作用14。我们的数据表明, 姜黄素不通过调节线粒体功能来增强胰岛素分泌。我们已经证明, 在可可儿茶单体的存在下, 培养出了线粒体电子传输链各组分的表达式5。我们还观察到增加呼吸在泄漏状态 (由治疗诱导的寡) 和与 ETS (电子运输系统, 诱导的 FCCP)。使用这一工具意味着未来研究的一些潜在目标。
作为替代胰蛋白酶去除细胞, 机械离解也可以执行使用提出的洗涤技术时, 完成在37° c。呼吸测量是在没有或存在姜黄素或可可儿茶单体 (图 4A-c) 的情况下进行的。这些数据表明, 观察到的趋势与胰酶制剂的细胞保持, 但灵敏度似乎减少。虽然控制和可可儿茶单体处理的细胞保持了葡萄糖刺激, 泄漏和 ETS 呼吸, 细胞处理的姜黄素只保留了葡萄糖刺激呼吸, 由于增加样本的变异性。虽然观察到的趋势与胰蛋白酶细胞的准备工作保持 (图 4D-f), 在所有参数的意义下降, 想必是更大的样本-样品变异引入机械离解和蛋白质浓度的正常化而不是细胞的数量。
这些数据表明, 该协议可用于测量完整的 INS-1 832/13 β细胞的呼吸。此外, 我们的协议提出了一个最佳的细胞数精确测量和一个优选的方法来准备细胞, 以最大限度地提高信噪比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57053/57053fig1.jpg"> 图 1: 具有代表性的832/13 β细胞的呼吸曲线.完整的832/13 β细胞为呼吸测量 (图 1A) 2.5 毫米葡萄糖, 16.7 毫米葡萄糖, 2.5 µM 寡, 2 µM FCCP (羰基氰化物-4-三氟甲氧基) 腙) 和2.5 µM Antimycin A。16.7 mM 葡萄糖部分 (图 1B) 的扩展显示了葡萄糖刺激呼吸的增加。以 1 x 106细胞/毫升的浓度测量细胞。漏指的是基底、nonphosphorylating 呼吸状态, ETS 指的是电子传输系统的不耦合呼吸能力, 而火箭则是指线粒体呼吸抑制后的残余氧消耗。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57053/57053fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57053/57053fig2.jpg"> 图 2: 测定葡萄糖诱发的呼吸测量所需的832/13 β细胞的数量.在 (A) 0.5 x 106单元/毫升 (B) 1 x 106单元/ml, 或 (C) 2 x 106单元/ml 中, 测量完整的832/13 β细胞的呼吸。(D) 与 0.5 x 106单元/ml、1 x 106单元/ml、2 x 106单元/ml 对应的蛋白质浓度。(E) 比较呼吸在 0.5 x 106细胞/毫升, 1 x 106细胞/毫升, 和 2 x 10 6 细胞/毫升.呼吸测量在2.5 毫米葡萄糖、16.7 毫米葡萄糖、2.5 µM 寡、2µM FCCP 和2.5 µM Antimycin A 条件下。呼吸作用以2.5 和16.7 毫米葡萄糖显示β细胞的葡萄糖被刺激的呼吸反应。呼吸作用在寡以后证明了漏呼吸作用 (基础, nonphosphorylating 呼吸状态)。FCCP 后的呼吸 (羰基氰化物-4-三氟甲氧基) 腙) 治疗表明 (电子运输系统的不耦合呼吸能力, ETS)。n = 6 独立运行。数据显示为平均± SEM. * p < 0.05, ** < 0.01, ** p < 0.001, *** p < 0.0001。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57053/57053fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57053/57053fig3.jpg"> 图 3: 使用胰蛋白酶释放后832/13 β细胞的呼吸测量.832/13 β细胞在单体可可儿茶或姜黄素存在或缺乏的情况下培养, 用胰蛋白酶释放细胞后进行呼吸测定。呼吸测量在2.5 毫米葡萄糖、16.7 毫米葡萄糖、2.5 µM 寡、2µM FCCP 和2.5 µM Antimycin A 条件下。呼吸与2.5 和16.7mM 葡萄糖证明葡萄糖被刺激的呼吸作用。呼吸作用在寡以后证明了漏呼吸作用 (基础, nonphosphorylating 呼吸状态)。FCCP 后的呼吸 (羰基氰化物-4-三氟甲氧基) 腙) 治疗表明 (电子运输系统的不耦合呼吸能力, ETS)。antimycin 治疗后的呼吸显示残余氧消耗 (火箭)。(A) 治疗832/13 细胞与姜黄素保持适当的变化, 呼吸由于治疗。(B) 有代表性的跟踪和 (C) 用姜黄素处理的细胞的平均数据表明, 832/13 β细胞呼吸没有变化。(D) 用可可儿茶单体处理832/13 细胞, 维持因治疗而引起的呼吸的适当变化。(E) 有代表性的跟踪和 (F) 可可儿茶单体处理细胞的数据平均值表明, 可可儿茶单体处理后所有参数的呼吸增加。(G) 将所有三参数一起提交以进行比较。n = 6 独立运行。数据显示为平均± SEM. * p < 0.05, ** < 0.01, ** p < 0.001, *** p < 0.0001。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57053/57053fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57053/57053fig4.jpg"> 图 4: 832/13 β细胞的呼吸测量在释放以后使用机械离解.832/13 β细胞在单体可可儿茶或姜黄素存在或缺乏的情况下培养, 用机械离解释放细胞后测定呼吸。呼吸测量在2.5 毫米葡萄糖、16.7 毫米葡萄糖、2.5 µM 寡、2µM FCCP 和2.5 µM Antimycin A 条件下。呼吸与2.5 和16.7 毫米葡萄糖显示β细胞的葡萄糖反应。呼吸作用在寡以后证明了漏呼吸作用 (基础, nonphosphorylating 呼吸状态)。FCCP 后的呼吸 (羰基氰化物-4-三氟甲氧基) 腙) 治疗表明 (电子运输系统的不耦合呼吸能力, ETS)。单元格 (A) 未经处理的细胞或细胞的平均数据 (B) 姜黄素或 (C) 可可儿茶单体证明机械离解在所有治疗中维持葡萄糖刺激的呼吸, 而泄漏和ETS 的意义仅在姜黄素治疗的细胞中丢失。未经处理的细胞与 (D) 姜黄素或 (E) 可可儿茶单体的比较表明, 可可儿茶单体在所有参数中的呼吸都有增加, 而姜黄素对呼吸的影响不大。(F) 将所有三参数一起提交以进行比较。n = 6 独立运行。数据显示为平均± SEM. * p < 0.05, ** < 0.01, *** p < 0.0001。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57053/57053fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds at JoVE.com

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

该协议的目的是测量葡萄糖介导的变化对线粒体呼吸的影响, 在天然化合物的存在, 在完整的832/13 β细胞使用高分辨率呼吸。

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

high-resolution-respirometry-to-measure-mitochondrial-function-intact

Nanyang Technological University

Research

JoVE Journal

Biology

11.8K 次观看.  Brigham Young University. 该协议的目的是测量葡萄糖介导的变化对线粒体呼吸的影响, 在天然化合物的存在, 在完整的832/13 β细胞使用高分辨率呼吸。

视频: High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

速度更快，高分辨率，MTPA-GFP基于线粒体融合含量的收购多个单元格的动力学数据，在并行使用共聚焦显微镜

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

科研

生物学

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

使用高解析度电脑断层可视化的三维结构和功能的植物血管

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解析度全山<em&gt;在原位</em杂交斑马鱼胚胎内，研究基因表达和功能

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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 ...

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.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

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

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Diagonal Method to Measure Synergy Among Any Number of Drugs

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many doses, allow sensitive measurement of drug interactions. However, these assays are costly and do not scale well for measuring interaction among many drugs. Several recent studies have reported drug interaction measurements using a diagonal sampling of the traditional checkerboard assay. This alternative methodology greatly decreases the cost of drug interaction experiments and allows interaction measurement for combinations with many drugs. Here, we describe a protocol to measure the three pairwise interactions and one three-way interaction among three antibiotics in duplicate, in five days, using only three 96-well microplates and standard laboratory equipment. We present representative results showing that the three-antibiotic combination of Levofloxacin + Nalidixic Acid + Penicillin G is synergistic. Our protocol scales up to measure interactions among many drugs and in other biological contexts, allowing for efficient screens for multi-drug synergies against pathogens and tumors.

In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations.

测量任意数量药物协同作用的对角方法

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.