我感谢，杰西卡Dominguez Rieg的博士批判性阅读这份手稿。支持这项工作是由美国心脏协会（科学家发展格兰特10SDG2610034），卡尔·戈特沙尔克研究资助的美国肾脏病学会，美国国立糖尿病，消化道和肾脏疾病奥布莱恩急性肾损伤研究中心（ P30DK079337）和退伍军人事务部。

1。解决方案<ol><li> 0.85％的氯化钠8.5克氯化钠/ 1负DDH 2 O（8.5克/升）。 </li><li> 0.5 mol / L的HEPES pH为7.4（在0.5 L的双蒸2 0，并调节pH值至7.4，用10 N的氢氧化钠溶解59.6克HEPES）。 </li></ol> 2。的FITC-菊粉注射液的制备<ol><li>称取FITC-菊粉制备5％的溶液和溶解在0.85％的氯化钠（通常为100毫克/ 2毫升0.85％的NaCl）中通过加热至90℃，直到完全溶解。 </li><li>称量溶解FITC-菊粉的解决方案，并注意重量。 </li><li>切20厘米一块透析膜（分子量截止:1000），并把在DDH 2 O 30分钟，以去除残留的膜和楠3冲洗几次之后。 </li><li>填写溶解FITC-菊粉透析膜和密封关闭。 </li><li>称量整个透析膜。 </li><li>将1升0.​​85％NaCl溶液中的透析膜，光在RO的24小时保护，并慢慢搅拌OM温度。 </li><li>整个透析膜的再次称重并计算新的浓度（水会渗透进入膜和未结合的FITC，或绑定FITC-菊粉&lt;1000分子量出膜，因此，FITC-菊粉的浓度会大幅下降） 。 </li><li> FITC-菊粉浓度（C）使用下列公式计算:C = N / V，其中n =的FITC-菊粉初始金额，V =新的量（重量透析管透析前后加上初始体积的差异FITC-菊糖溶液）。 </li><li>在使用前通过0.22μm的注射器过滤器，透析的FITC-菊糖溶液通过过滤消毒。 </li><li>不断FITC-菊粉保护光（铝箔），在4℃下渗析和消毒的FITC-菊粉可用于长达2周。参加FITC-菊粉可以再加热溶解，在90℃下为几分钟。 </li></ol> 注意:如果使用FITC-sinistrin 16-18，这并不需要透析，透析所需的所有步骤可以跳过。 3。注射和采血车<ol><li>以体重（BW）的小鼠实验前。 </li><li>简单异氟醚麻醉小鼠。 </li><li>对于高吞吐量的6只小鼠，请按照图3中提供的协议。 </li><li>使用G30半针（除去气泡从100微升汉密尔顿注射器首先使用G26半针，然后切换到G30半针，注入2微升/ g体重透析的FITC-菊粉到球后神经丛24注射）。 </li><li>鼠标尾部用剪刀剪下1毫米的一次采集血液3，5，7，10，15，35，56和75分钟后钠肝素minicapillaries中注入。采血后CHA密封的密封minicapillaries。 </li><li>保持避光样品。 </li><li>将密封minicapillaries里面造血暴击毛细血管和离心5分钟。 </li><li>通过使用金刚石切割器，并通过移液到0.2毫升管中转移整个等离子minicapillaries额。 </li><li> 1:10稀释，使用2微升血浆和18微升0.5摩尔/ L HEPES（pH7.4）中，在新的0.2毫升管（样品贮存过夜，在4℃下进行荧光测量）。 </li><li>测量稀释样品2微升的NanoDrop 3300重复。的描述和使用的仪器是描述在这里5。 </li></ol> 4。配制标准<ol><li>将血液收集在相同的背景应变肝素涂层的血细胞比容毛细管老鼠，降速，用0.5 mol / L的HEPES（pH 7.4）中稀释1:10，通常为80μl血浆+ 720微升HEPES。 </li><li> FITC-菊粉注射液稀释的小鼠血浆稀释，得到以下的标准稀释: 1:10:10微升未稀释的FITC-菊粉溶液+ 90微升稀释鼠标的血浆 1:100:10亩，L（1:10）溶液+ 90微升稀释鼠标的血浆 1:1000:10微升（1:100）溶液+ 90微升稀释鼠标的血浆 1:2,000:30微升（1:1）溶液+ 30微升稀释鼠标的血浆 1:10,000:10微升（1:5）溶液+ 40微升稀释鼠标的血浆 1:20,000:20μL（1:1）溶液+ 20微升稀释鼠标的等离子 1:100000:10微升（1:5）溶液+ 40微升稀释鼠标的血浆 标准的浓度将取决于透析，将永远是不同的各制剂的FITC-菊粉。这是必须输入的浓度标准曲线每次新。 </li><li>分析数据计算肾小球滤过率与通过使用适当的软件（ 例如 GraphPad棱镜）这里15，20中所描述的和所示的代表结果的第一个两阶段的指数衰减函数。 </li></ol>

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笔者收到的资金，以支付本出版物由赛默飞世尔科技。

本研究中描述了一种技术，在有意识的小鼠的肾小球滤过率的测定由单一大剂量注射和8的血液样本中的荧光分析，通过两相指数衰减。 1鼠标的GFR测量需要75分钟。通过使用一个特殊的流程图，它是能够测量在130分钟6小鼠的肾小球滤过率。重复此流程图协议是可行的，每天1-4次，并测量在24只小鼠的肾小球滤过率。我们修改了这个方法，因此，它有可能从一条小尾巴剪断，而不是尾静脉穿刺，这是更快，更容易调查执行相比，尾静脉穿刺取血。通过使用10微升的血细胞比容毛细管和1:10稀释，这是可能的，以减少总的所需要的血液量&lt;100微升。最后，荧光测量使用的NanoDrop 3300的fluorospectrometer，它允许在1-2微升稀释样品的荧光测定。此方法将得到感兴趣的调查官员岭真GFR值。相比之下，在小鼠体内的肌酐测量有一些弱点，包括"肌酐盲区"和困难相关的特异性和敏感性测定。 "肌酐盲区范围"限制了它的功能作为GFR的标记物，因为血清肌酐保持在正常范围内，直到50％的肾功能丢失19。由于一些研究者利用他们的研究转基因小鼠肾脏生理和病理生理学研究，承担以下问题:假设一个基因敲除小鼠基因X将有一个显着降低肾小球滤过率（40％）的野生型小鼠相比，通过比较简单的筛选血浆肌酐不会检测到这种差异。需要有一个良好的GFR测定准确的方法变得更为关键的肾小球滤过率的差异较小成为。 老鼠，非肌酐chromagens的高浓度，在他们的血浆和尿中的干扰与市售可用肌酐检测。作为一个结果，这是基于测定碱性苦味酸盐Jaffe反应进行高估肌酐在血浆中浓度相比，用高效液相色谱法（HPLC）测定6，13。另一个限制是，血清肌酐低于检测限相比，高效液相色谱法测定的一些市售的检测。然而，通过酶促反应调查能够确定血浆肌酐在小鼠10，22。 GFR在类似的范围内被发现在清醒小鼠使用可比单推注方法以及尿FITC-菊粉清除14。在他们的协议相比，我们的协议，使用尾剪断并不需要特别的预防措施，以保持血管的完整性相比，尾静脉或大隐静脉穿刺。 ，对于尿/等离子为基础的FITC-菊粉清除有必要手术植入两个渗透英里nipumps。有两个泵是要达到足够高的稳态等离子体FITC-菊粉浓度从背景有明显区别。这也需要使用一个完整的，定时的尿液收集代谢笼。网箱需要清洗占25-37％FITC-菊粉，不漂洗，否则发生的损失。 使用此FITC-菊粉方法的缺陷是有限的。主持人应注意下列:（一）保证有注射器用于注射的FITC标记的溶液中没有气泡，（ⅱ）的完整的球后注射必须被实现，因为注射量确定计算肾小球滤过率，及（ ⅲ）小鼠将最有可能在75分钟GFR实验溲，因此可避免污染的血液样本的尿液，这将产生一个非常高的FITC-菊粉的浓度为肾脏浓缩能力的结果。 一般优势Ø上本FITC-菊粉的方法，包括以下内容:（一），（ⅱ）有可能多次重复测量（iii）该法可在有意识的小鼠，进行相同的鼠标，它是一个非放射性技术（ iv）概无需要收集尿液，及（v），可以利用高通量选项。为单一的大剂量注射的缺点包括需要为FITC-菊粉透析，这是可以克服的使用FITC标记，FITC-sinistrin，这是现在使用一个特殊的小型化的移动设备18可以测量皮。即使肾小球滤过率的测量，这是不可能用这种方法测量流体或离子排泄分数。 两者合计，该方法提供了一种高通量的方法，测量在有意识的小鼠的肾小球滤过率的可行性。因此，这种技术可能感兴趣的其他调查那些有兴趣在确定肾功能的影响。

汇率形成血浆超滤液上面的肾小球已成为用于评估肾功能（肾小球滤过率，肾小球滤过率）的基础。 GFR理想的标记，以确定应自由过滤，既不分泌或重吸收和代谢。这样的行为被认为是真正的菊粉，菊粉清除已成为金标准测定肾小球滤过率3。肌酐清除率，而被广泛使用作为衡量在人类和动物的肾小球滤过率，肌酐不能满足这个要求的一个理想的GFR标记。人类的肌酐清除率大约10 - 40％的后果是肾小管主动分泌1，11，并在小鼠和大鼠4，7，9，22，肌酐是肾分泌也很突出。肾功能不全患者的肾小管分泌肌酐，这就限制了它的值作为GFR 1，2的标志物是已知会增加。我们先前已经表明，有机阴离子转运亚型有助于3（OAT3）小鼠肾肌酐分泌22。然而，菊粉清除率通常需要使用放射性标记的菊粉（3 H或14 C）。可使用的放射性测定菊粉在麻醉和有意识的小鼠，但藏着众多的安全规章和严格的处理问题，与使用放射性同位素的固有的缺点。沿着这些线路，手术间隙制剂测定肾小球滤过率的耗时以及终端的性质。 1999年洛伦兹等人 12介绍FITC-菊粉麻醉小鼠单肾GFR的标志物。五年后，齐等15确定的全肾GFR在清醒小鼠使用FITC-菊糖。现在替代协议使用FITC-菊粉作为外生光标测量GFR在清醒和麻醉小鼠。这些协议中保留的放射性菊糖的灵敏度通过使用荧光detecti，和绕过的缺点，使用放射性标记的标记。 FITC-菊粉法修改由其他7，8，22，23，利用微量的NanoDrop 3300 fluorospectrometer能力。这可以降低总样本量&lt;100微升血每GFR测量所需。 随着本出版物中提供的方法和高吞吐量的协议，我们展示了24意识的小鼠每天测量GFR的可行性。其他研究者研究小鼠肾小球滤过率，这种技术可能是有用的，我们希望这将取代不太准确的方法，肾功能，如肌酐和尿素氮指数。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FITC-inulin</td>
			<td>Sigma-Aldrich</td>
			<td>F3272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H7706</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Webster Veterinary</td>
			<td>78366551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dialysis membrane (MWCO 1000)</td>
			<td>Spectrum Laboratories</td>
			<td>132636</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Membrane closure</td>
			<td>Spectrum Laboratories</td>
			<td>132736</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>80 &mu;l Hematocrit capillaries</td>
			<td>Drummond Scientific</td>
			<td>22362566</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hematocrit centrifuge</td>
			<td>IEC</td>
			<td>Micro-MB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 &mu;l Na+-Heparin minicaps</td>
			<td>Hirschmann</td>
			<td>9000210</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe (100 &mu;l)</td>
			<td>Hamilton</td>
			<td>80601</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorospectrometer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>NanoDrop 3300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter (0.22 &mu;m)</td>
			<td>Spectrum Laboratories</td>
			<td>880-26464-UE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sealant</td>
			<td>Ch&acirc;-seal</td>
			<td>510</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needles (G26 &frac12; and G30 &frac12;)</td>
			<td>Becton Dickinson</td>
			<td>305111 &amp; 305106</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a high-throughput method for measurement of glomerular filtration rate in conscious mice

肾小球滤过率（GFR）的测量是肾功能评估的黄金标准。目前，研究确定GFR测量的内源性生物标志物的肌酐或外源性应用放射性标记的菊粉（3 H或14 C）的水平。肌酐〜50％，因此总肾功能肌酐排泄肌酐，近端肾小管分泌账户的主要缺点是不是一个可靠的GFR标记。根据上进行的实验中，菊粉清除率，可通过静脉注射单一剂量注射或连续输注（静脉或渗透微型泵）。两种方法都要求收集的血浆或血浆和尿液。放射性标记的菊粉的其它缺点包括使用同位素，耗时的手术准备的动物，和要求的终端实验。在这里，我们描述了一种方法，它使用一个单一的静脉注射异硫氰酸荧光素（FITC）标记的菊粉和1-2微升稀释的血浆中测量其荧光。通过施加二室模型，8每只小鼠的血液采集，它是可以测量肾小球滤过率的24只小鼠，每天使用一种特殊的工作流协议。此方法只需要短暂的异氟醚麻醉与所有的血液样品被收集在一个非受限制和清醒鼠标。另一个优点是，它有可能超过一段时间的数个月，处理（ 即进行配对实验饮食结构的改变或药物应用）按照小鼠。我们希望，这种技术是非常有用的测量GFR研究小鼠肾脏功能的其他研究者，将取代不太准确的方法，估计肾功能，如血浆肌酐和尿素氮。

肾小球滤过率将使用下面的公式计算: GFR = N /（A/K1 + B/K2），其中 N =注入量（N = C•V，C = FITC-菊粉浓度，V =注入量） A = SPAN1时，y轴截距消除 K1 =衰变常数消除 B =跨度2，y轴截距分布 K2 =衰变常数为分配 注意:甲，乙，K1和K2是GraphPad棱镜，每一个软件能够分析两相呈指数衰减的缩写都可以使用，但是，其他的科学软件可能使用不同的缩写。 通过采用一个两阶段的指数衰减曲线拟合，这是基于在血浆中FITC-菊粉的浓度，在图1中所示的曲线与应获得。由上面所示的式（计算肾小球滤过率，在这里所描述&lt;SUP&gt; 20）可以发现在小鼠的1型糖尿病（ 图2），检测肾功能15或肾小球滤过8,23减值。 <img alt="图1" src="/files/ftp_upload/50330/50330fig1.jpg" /> 图1。来衡量GFR清醒小鼠血浆中动力学单剂量静脉注射FITC-菊粉是用来计算肾小球滤过率，二室模型。在双室模型，初始的快速衰减阶段表示FITC-菊粉从血管内室的细胞外液中的再分配。从血浆中后期阶段，在FITC-菊粉浓度的衰减越慢，主要反映全身清除。 <img alt="图2" src="/files/ftp_upload/50330/50330fig2.jpg" /> 图2。 Detection的野生型小鼠糖尿病高滤过，I型糖尿病引起的腹腔应用链脲佐菌素（STZ，50毫克/公斤，连续5天）。 GFR测定（基础）和5周后诱导的糖尿病。由于管球反馈，近端重吸收增加导致糖尿病早期高滤过21是重现这一发现在野生型小鼠组（n = 10，P &lt;0.01基底相同的鼠标）。 <img alt="图3" src="/files/ftp_upload/50330/50330fig3.jpg"/> 图3。在一组6只小鼠的肾小球滤过率的测量为了测量整个肾脏的肾小球滤过率的单一大剂量注射法的流程图 ，8个血液样品必须被收集在3，5，7，10，15，35，56和75分钟后FITC-菊粉注射。数字表示的时间点。括号中的数字表示样本数。时间点的垂直红线表示注射时间点（在0，11，22，33，44和55分钟）。每只小鼠的标记具有不同的颜色。为了获得连续的血液采集，按照箭头。灰色框表示下鼠标时，异氟醚麻醉注射前必须准备。使用此协议2个血液采集来自不同小鼠之间的最短时间是1分钟。

Watch this Scientific Journal Video about A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice at JoVE.com

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

肾小球滤过率（GFR）是测量评估肾功能的金标准。在这里，我们描述了一种高通量的方法，该方法允许由一个两阶段的指数衰减模型，通过使用一个单一的大剂量注射，测定异硫氰酸荧光素（FITC）在血浆中菊粉和肾小球滤过率的计算肾小球滤过率的测定在有意识的小鼠。

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

a-high-throughput-method-for-measurement-glomerular-filtration-rate

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

43.2K 次观看.  University of California, San Diego. 肾小球滤过率（GFR）是测量评估肾功能的金标准。在这里，我们描述了一种高通量的方法，该方法允许由一个两阶段的指数衰减模型，通过使用一个单一的大剂量注射，测定异硫氰酸荧光素（FITC）在血浆中菊粉和肾小球滤过率的计算肾小球滤过率的测定在有意识的小鼠。

视频: A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

高通量测量和环境样品中的有机磷分类

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

冰帽:为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

生理学实验室演示:肾小球滤过率在一个老鼠

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

高通量清醒小鼠肾小球滤过率的测定方法

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