这项研究得到了韩国科学和技术研究所校内研究补助金 (2E27513) 和农业、粮食和农村事务部 (315067-03) 资助的高增值食品技术发展方案 (IPET) 的支持。

注意: 整个实验必须在20和 #176 保持的干净的隔离实验室中进行; 在蠕虫和细菌处理过程中, 低粉尘和最小污染的 C。为此, 必须在酒精灯的火焰或使用干净的长凳上进行实验. 
 1. 维护 c. 线虫 和鸡蛋准备化学测试 <ol> <li> 维护 线虫 N2 (var) 在 NGM 琼脂板上喂养的活 大肠杆菌 OP50 在20和 #176; C 12 , 13 . </li>
	<li> 使用漂白剂溶液处理的龄期同步卵 5 或时间蛋铺设方法 6 14 , 15 
	注: 在准备鸡蛋之前, 用热灭活的 大肠杆菌 OP50 涂依托含 NGM 板, 如下所述。热灭活的 大肠杆菌 OP50 用于将 大肠杆菌 6 、 16 的新陈代谢活动降至最低。
</ol> 2。制备热灭活的 大肠杆菌 OP50 在毒性试验中, 为饲料中的 线虫 <ol> <li> 准备500毫升的双酵母胰 (戴阳天) 培养基, 如表1所述, 并接种单一菌落的 大肠杆菌 OP50 培养Luria-Bertani (LB) 琼脂平板 5 . </li>
	<li> 孵育 大肠杆菌 OP50 在14小时的震动孵化器中, 37 和 #176; C 和 150 rpm. </li>
	<li> 离心30分钟后在 3220 x g 和 20 #176; C、取出所有上清戴阳天培养基, 并添加混25毫升的 s-缓冲器、250和 #181; 1 米 MgSO 4 , 25 和 #181; 5 毫克/毫升胆固醇 (溶于乙醇)。所有这些解决方案都是无菌的. </li>
	<li> 重细菌并将其转移到50毫升的一次性塑料管中. </li>
	<li> 热失活, 孵育悬浮 大肠杆菌 OP50 在水浴30分钟, 在65和 #176; C. </li>
	<li> 冷却到室温并存储在4和 #176; C 直到使用。这可以存储长达一个月. </li>
</ol> 3。制备含有1% 二甲基亚砜或750和 #181 的 NGM 板; M 依托 <ol> <li> 准备两个干净的200毫升玻璃瓶。加入0.6 克氯化钠, 0.5 克蛋白胨, 3.4 克琼脂, 195 毫升蒸馏水, 一瓶磁力搅拌器。在另一个空瓶子里加一个磁力搅拌器. </li>
	<li> 将这两个瓶子在121和 #176 上压釜15分钟, 然后在水浴中冷却30分钟, 在55和 #176; c. </li>
	<li> 添加 0.2 mL 1 米 CaCl 2 , 0.2 毫升的5毫克/毫升胆固醇, 0.2 毫升 1 m MgSO 4 , 5 毫升 1 m KPO 4 (所有这些解决方案是无菌), 然后混合他们的磁搅拌在热板上, 在55和 #176; C. </li>
	<li> 将混合 NGM 介质分成两个相同体积的瓶子 (每瓶100毫升)。然后, 将1毫升的亚甲基亚砜添加到一个瓶子中, 再用磁力搅拌再混合。将另一个瓶子放在55和 #176 的水浴里, 直到下一步。分3毫升的含亚砜的培养基, 每 35 x 10 mm 2 培养皿。从这一步可以制成大约33含亚砜的 NGM 板. </li>
	<li> 混合其他瓶子使用磁性搅拌, 添加1毫升的75毫米依托 (股票溶解在二甲基亚砜), 再次混合, 并重复分程序 (步骤 3.4)。从这一步中可以得到大约33依托 (750 和 #181; M)-包含 NGM 板. 
	注意: 在相关的前一篇文章 6 中, 我们测试了0、250、500和1000和 #181; 依托。根据这一数据, 本文选取了 750 #181; 依托剂量. </li>
	<li> 将含有化学成分的 NGM 板冷却至室温, 将其置于室温下约3小时, 并将其存储在4和 #176; C 直到使用. 
	注意: 在光敏化学品的情况下, 阻挡光, 以尽量减少光分解. 
	注: 可选, 做一个正常的 NGM 板没有化学品的鸡蛋铺设化验, 可以执行使用两种不同的化学处理条件如下所述. </li>
	<li> 添加100和 #181; 热灭活 大肠杆菌 OP50 (如2节所述) 并在每个 NGM 板上传播。允许在20和 #176 的 c 线虫 孵化器中过夜, 用于化学测试. </li>
</ol> 4。测量 C. 线虫 增长 <ol> <li> 将年龄同步的 线虫 的卵转移到 NGM 板上, 再配以1% 亚砜或750和 #181; 我依托. </li>
	<li> 孵育 c. 线虫 在20和 #176 的孵化器中; c 为4天。用立体显微镜观察蠕虫, 每天拍摄显微图像。通常20X 放大 (1X 物镜, 2X 变焦放大, 和10X 目镜镜头) 是适合的增长观察的 C. 线虫 。还采取显微镜阶段千分尺的显微图像的蠕虫大小校准. 
	注意: 饥饿影响毒性试验。因此, 饲料有足够的细菌或调整和 #160; 在开始时转移了 C. 线虫 卵数。观察到3天, 排除饥饿的可能性. </li>
	<li> 使用 ImageJ 软件在每个时间点测量用亚砜或依托处理的 C. 线虫 的体长. 
	注: 对于每个样本测量, 50 蠕虫是足够的统计分析。
	<ol> <li> 在 ImageJ 中, 打开显微镜阶段千分尺图像 (#39; 文件和 #39; #8594; #39; 打开和 #39;). 
		注: 千分尺图像和蠕虫图像 (步骤 4.2) 必须是相同的放大倍数. </li>
		<li> 通过使用和 #39、直线和 #39; 工具, 在千分尺校准图像上拖动1000和 #181;
		<li> 单击和 #39; 分析和 #39; #8594; #39; 设置规模和 #39; 输入1000和 #181; 我进入和 #39; 已知的距离和 #39; 框和 #39; 长度和 #39 的单位; 框, 分别。检查和 #39; 全局和 #39; 单击并 #39; 确定 #39;. </li>
		<li> 打开蠕虫图像 (#39; 文件和 #39; #8594; #39; 打开和 #39;)。使用和 #39; 手绘线和 #39; 工具, 沿每个蠕虫的特征绘制一条线。通过单击和 #39 获取正文长度数据; 分析和 #39; #8594; #39; 测量和 #39;. </li>
		<li> 对50蠕虫重复这些步骤. </li>
	</ol> </li> </ol> 5。 C. 线虫 产卵试验 <ol> <li> 在包含亚甲基亚砜或依托的 NGM 板上孵育年龄同步的卵, 在64小时 (第一次出生之前), 直到20和 #176 的年轻成人阶段; c. 
	注意: 在生长测量实验中使用蠕虫是可选的。这是方便做的生长测量和鸡蛋铺设化验一起. </li>
	<li> 将5只成年蠕虫转移到新的无化学品的 NGM 板 (条件 1, 在一定时间内进行化学处理, 从卵到幼成体阶段) 或新的 NGM 板含有相同的化学预处理 (条件 2, 连续曝光化学品通过整个实验期), 然后孵化为24小时. 
	注意: 建议每种治疗方法使用 3-5 NGM 板进行复制;这些复制可用于统计分析. </li>
	<li> 转移到新的 NGM 板如上所述, 每天计数的鸡蛋数。计数蠕虫爬行, 死亡, 并在内部孵化ed. </li>
	<li> 重复这些步骤, 直到蠕虫不再下蛋, 通常在5天内. </li>
	<li> 计算每个盘中每条蠕虫的产卵数, 并在5天内每天对这些值求和, 以确定产卵的总数量. 
	注意: 排除从计算中抓取和内部孵化的蠕虫的数量. </li>
</ol>

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作者没有什么可透露的。

在这篇文章中, 我们描述的毒性评价的化学品在线虫, 土壤线虫, 使用依托作为毒物的例子。为此, 我们使用了两个实验条件。在第一个集合中, C. 线虫生长在含有从卵到幼成虫阶段的依托板上, 然后允许蠕虫在正常的 NGM 板上产卵, 而不使用化学物质。在第二个实验组中, 在整个实验期间, 用依托连续处理线虫。通过比较两个实验条件下的卵数, 我们可以确定被测化合物的生殖毒性...

毒理学评价对制药、营养和妆的发展以及各种环境毒素的风险评估都是必不可少的。啮齿类动物模型是其中一个最普遍的在体内实验系统为这毒理学研究;另外, 哺乳有机体, 如C. 线虫也被广泛使用。非哺乳动物毒理学评价模型是有益的, 因为不仅动物伦理问题, 而且它们的方便性和实用性考虑 cost-effectiveness, 可维护性, 速度和重现性1,2 ,3,4。
线虫是一种土壤圆形蠕虫, 在各种基础和应用生物学和化学研究中被作为动物模型来开发。它是一个1毫米长, 透明的线虫, 这是简单地维持在固体或液体线虫生长培养基 (NGM) 喂养的细菌菌株大肠杆菌OP50。c. 线虫的生命周期较短, 野生类型的 N2 c. 线虫约有300只卵。因此, 它很容易被传播到用作实验材料3,4,5。线虫在许多药物和环境污染物的毒理学研究中也得到了广泛的应用6,7,8,9。
由于许多抗癌药物的目标是迅速分裂的癌细胞, 他们也可以损害迅速分裂正常细胞, 如骨髓, 肠上皮, 毛囊细胞。例如, 拓扑抑制抗癌药物靶向癌细胞的 DNA 复制过程;因此, 它们也抑制了快速分裂正常细胞。每一个生物体都有 topoisomerases, 而这些拓扑抑制剂最有可能影响环境生态系统6,10,11。因此, 使用模型动物的药物毒理学评价平台对药品的开发和环境风险评估都是有价值的。
在本文中, 我们描述的详细的协议, 以测试毒性的依托, 这是一个临床抗癌剂的目标拓扑 II, 作为一个模型有毒化学品在线虫。为此, 我们将描述的测量方法的身体大小和总数量的鸡蛋奠定了在线虫处理的依托。

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Affymetrix, USA</td>
			<td>10906</td>
			<td></td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans N2</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>Wild type</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma, USA</td>
			<td>C3045</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma, USA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli OP50</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>Sigma, USA</td>
			<td>E1383</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software (ver 1.4)</td>
			<td>Natinoal Institute of Health, USA</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Jenopitk, Progress Gryphax, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck, USA</td>
			<td>107213</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#215; 10 mm Petri dish</td>
			<td>SPL Life Sciences, South Korea</td>
			<td>10035</td>
			<td></td>
		</tr>
		<tr>
			<td>90 &#215; 15 mm Petri dish</td>
			<td>SPL Life Sciences, South Korea</td>
			<td>10090</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Nikon, Japan</td>
			<td>SMZ800N</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Becton Dickinson, USA</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effect of chemicals on the growth and reproduction of caenorhabditis elegans

毒理学评价对于了解化学物质对生物科学在基础和应用生物学领域的影响至关重要。哺乳土壤圆形蠕虫,线虫线虫, 是一个有价值的模型生物毒理学研究, 因为它的方便和缺乏动物伦理问题与哺乳动物的动物系统相比。在本议定书中, 详细介绍了在线虫中对化学品进行毒理学评价的过程。一种临床抗癌药物, 依托, 它的目标人类拓扑 II 和抑制 DNA 复制的人癌细胞, 被选为一个模型试验化学。年龄同步的线虫卵被暴露在二甲基亚砜 (亚砜) 或依托, 然后通过立体显微镜观察, 每天4天监测c. 线虫的生长情况。用依托和亚甲基亚砜处理的从线虫中得到的卵总数也用立体显微镜计算。依托治疗显著影响了线虫的生长和繁殖。通过对不同药剂处理期的蚯蚓卵总数的比较, 可以断定, 化学物质对线虫繁殖的生殖毒性是可逆的或不可逆转的。这些协议可能有助于发展各种药物和风险评估的环境毒物。

依托 (24-96 h) 的治疗明显延缓了线虫的生长。经过 96 h 的孵化, 依托处理的蠕虫生长到0.86 毫米的体长, 而车辆处理的蠕虫增长到1.04 毫米 (图 1)。在立体显微镜观察下, 生长迟缓也有明显的观察 (图 2)。我们开始在72小时的孵化中看到由汽车处理过的蠕虫的卵。另一方面, 在96小时的依托治疗后观察卵子。从这些数据, 我们推?...

Watch this Scientific Journal Video about Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans at JoVE.com

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

一个基本的协议, 以评估化学品的毒性在模型动物,线虫线虫, 描述。该方法对药品的开发和各种环境污染物的风险评估都具有很好的实用价值。

测定化学物质对线虫线虫生长和繁殖的影响

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

measuring-effect-chemicals-on-growth-reproduction-caenorhabditis

Research

JoVE Journal

Medicine

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

16.8K 次观看.  Korea Institute of Science and Technology. 一个基本的协议, 以评估化学品的毒性在模型动物,线虫线虫, 描述。该方法对药品的开发和各种环境污染物的风险评估都具有很好的实用价值。

视频: Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Measuring the Strength of Mice

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score maximum on this task; it is a quick but insensitive gross screen, and the weights test described in this article will provide a finer measure of muscular strength.
There are also several strain gauge-based pieces of apparatus available commercially that will provide more graded data than the inverted screen test, but their cost may put them beyond the reach of many laboratories which do not specialize in strength testing. Hence in 2000 a cheap and simple apparatus was devised by the author. It consists of a series of chain links of increasing length, attached to a "fur collector" a ball of fine wire mesh sold for preventing limescale build up in hard water areas. An accidental observation revealed that mice could grip these very tightly, so they proved ideal as a grip point for a weight-lifting apparatus. A common fault with commercial strength meters is that the bar or other grip feature is not thin enough for mice to exert a maximum grip. As a general rule, the thinner the wire or bar, the better a mouse can grip with its small claws.
This is a pure test of strength, although as for any test motivational factors could potentially play a role. The use of scale collectors, however, seems to minimize motivational problems as the motivation appears to be very high for most normal young adult mice.

Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as coordination.

强度测量小鼠

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score ...

科研

医学

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1（PD-L1）免疫组化染色石蜡的嵌入式幻灯片的胰腺癌组织

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

在感染艾滋病毒的个体的测量弱者。体弱患者的识别是第一步改良与弱者拨回

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

The Influence of Liver Resection on Intrahepatic Tumor Growth

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not detectable by routine clinical imaging, may be stimulated by hepatic regeneration factors after liver resection. It is not entirely clear, however, which factors are crucial for tumor recurrence.
The presented mouse model may be useful to explore the mechanisms that play a role in the development of recurrent malignant lesions after liver resection. The model combines the easy-to-perform and reproducible techniques of defined amounts of liver tissue removal and tumor induction (by injection) in mice. The animals were treated with either a single laparotomy, a 30% liver resection, or a 70% liver resection. All animals subsequently received a tumor cell injection into the remaining liver tissue. After two weeks of observation, the livers and tumors were evaluated for size and weight and examined by immunohistochemistry.
After a 70% liver resection, the tumor volume and weight were significantly increased compared to a laparotomy alone (p &#60;0.05). In addition, immunohistochemistry (Ki67) showed an increased tumor proliferation rate in the resection group (p &#60;0.05).
These findings demonstrate the influence of hepatic regeneration mechanisms on intrahepatic tumor growth. Combined with methods like histological workup or RNA analysis, the described mouse model could serve as foundation for a close examination of different factors involved in tumor growth and metastatic disease recurrence within the liver. A considerable number of variables like the length of postoperative observation, the cell line used for injection or the timing of injection and liver resection offer multiple angles when exploring a specific question in the context of post-hepatectomy metastases. The limitations of this procedure are the authorization to perform the procedure on animals, access to an appropriate animal testing facility and acquisition of certain equipment.

A high incidence of tumor recurrence after resection of liver metastases remains an unsolved problem. The illustrated mouse model may be useful to investigate the reasons for such recurrences. It combines a liver resection model with intrahepatic tumor cell injection for the first time.

肝切除肝内肿瘤生长的影响

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

比浊法对人洗涤血小板:所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

测定细菌和化学物质对开牛结不二病的肠道渗透性的影响

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Quantification of Optic Nerve Cross Sectional Area on MRI: A Novel Protocol using Fiji Software

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic nerve cross-sectional assessment and quantification using 3 T MRI for image acquisition and ImageJ's Fiji software for image processing quantification. Image acquisition was performed using 3 T MRI, with proper instructions for the patient to ensure straight fixation during imaging. A T2-weighted fat suppressed sequence was used. A coronal cut taken 3 mm behind the globe and perpendicular to the optic nerve axis should be uploaded to the software. Using the threshold function, the white matter area of the optic nerve is selected and quantified, thus, eliminating inter-individual measurement bias. We also described the normal limits for the optic nerve cross-sectional area according to age, based on previously published literature. We used the described protocol to assess optic nerve of a suspected glaucoma patient. The optic nerve cross-sectional area was found to be within the normal limits, a finding further confirmed via optical coherence tomography of the optic nerve.

We provided a detailed protocol for a standardized method of optic nerve assessment and quantification using MRI, utilizing a widely available imaging sequence, and open access software for image analysis. Following this standardized protocol would provide meaningful data for comparison between different patients and different studies.

MRI 上光学神经横截面区域的量化:使用斐济软件的新协议

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic ...