我们想感谢 Gráinne 的编辑协助。

外周血和血清被收集和储存的健康捐赠者和患者谁给予书面知情同意参与该项研究。该研究议定书是根据《1964赫尔辛基宣言》规定的道德标准, 由地方道德委员会批准的。
1. 星展集团的血液贮存
<ol><li>
采血样本收集
	<ol>
<li>收集3毫升的外周血样本在3毫升管与5.4 毫克乙二胺乙酸 (EDTA)。 注: 在8小时内尽快将外周血储存在 DBS 内。</li>
		<li>在保护卡的右下角写上病人代码号。</li>
		<li>小心地用5毫升的吸管并用重悬血液。</li>
	</ol>
</li>
	<li>
星展的血液斑点
	<ol>
<li>吸管和转移一点血液 (50 µL) 到保护卡 (材料表) 与50–200µL 提示。</li>
		<li>重复步骤1.2.1。有一共有3–5的血点。扔掉旧的小费, 并采取一个新的每一个血液整除。</li>
		<li>让星展集团在室温下过夜, 在一个开放的不吸水表面, 避免阳光直射的水平位置。把保护卡的盖子打开, 方便血液干燥。 注: 在干燥过程中, 空气或粉尘中的任何微粒都不会影响下游的应用。</li>
		<li>将卡在室温下存放在带有干燥剂的塑料袋中, 直到使用。</li>
	</ol>
</li>
</ol>2. dna 提取试剂盒 (用于干血斑点切片) 从 DBS 开始
<ol><li>
样品准备
	<ol>
<li>使用卡上的3个干血点。从卡上切下每个干血点, 并用镊子将它们与卡分开。将分离的干血点切成小块 (直径约1毫米)。</li>
		<li>将小块放入1.5 毫升离心管中。</li>
	</ol>
</li>
	<li>
样品消化
	<ol>
<li>将试剂盒 (材料表) 中提供的180µL 裂解缓冲液1添加到管中。添加20µL 蛋白酶 K。不要将裂解缓冲剂1与蛋白酶 k 混合, 然后再用血斑点卡吹打到1.5 毫升管。混合涡流。</li>
		<li>把管子放在 thermoincubator 中, 在56摄氏度处孵化60分钟。如果 thermoincubator 没有配备摇床, 则每10–15至少十年代将样品涡流, 注意不要让样品的温度降低。简单地离心管子从盖子去除下落。</li>
	</ol>
</li>
	<li>
洗涤步骤
	<ol>
<li>在十年代加入200µL 溶解缓冲 2 (材料表) 和漩涡。</li>
		<li>将管子放在 thermomixer 或加热的轨道孵化器中, 在70摄氏度处孵化10分钟。如果 thermoincubator 没有配备振动筛, 则至少在十年代将样品涡流一次, 注意不要让样品的温度降低。</li>
		<li>重复步骤2.3.1。将400µL 的裂解物从1.5 毫升管转移到洗脱柱 (材料表)。离心柱在 6000 x g 1 分钟。</li>
		<li>将洗脱柱放入新的2毫升收集管中, 丢弃旧的。</li>
		<li>添加500µL 洗涤缓冲 1 (材料表) 和离心机在 6000 x g 为1分钟。</li>
		<li>将该列放入一个新的2毫升收集管并丢弃旧的。</li>
		<li>添加500µL 洗涤缓冲 2 (材料表) 和离心机在 6000 x g 为1分钟。</li>
		<li>将该列放入一个新的2毫升收集管并丢弃旧的。</li>
		<li>离心机在 2万 x g 3 分钟干燥膜。</li>
	</ol>
</li>
	<li>
洗脱步骤
	<ol>
<li>将该柱放入新的1.5 毫升离心管中, 并丢弃收集管。</li>
		<li>将50µL 蒸馏水放到膜的中心。室温下孵育10分钟。</li>
		<li>离心管在 2万 x g 1 分钟收集洗脱液并再次放置在膜的中心。</li>
		<li>离心管在 2万 x g 1 分钟丢弃膜。</li>
		<li>使用分光光度计测量 DNA 浓度。</li>
		<li>储存的 DNA 在-20 摄氏度, 直到使用。 注: 从星展中提取的 DNA 可以用来进行几种分析, 如 SNP 评估的实时 PCR 或 DNA 测序。</li>
	</ol>
</li>
</ol>3. 用于 DSS 的血清贮存
<ol><li>
采血样本收集
	<ol>
<li>收集7毫升的外周血样本在7毫升管没有 EDTA。让血样在室温下凝固30分钟。</li>
		<li>在保护卡的右下角写上病人代码号。</li>
		<li>在室温下离心 980 x g的血液样本15分钟。 注: 在8小时内尽快将血清作为 DSS 贮存。</li>
		<li>小心地从试管中取出上清, 将其转移到新的15毫升管上。小心地用5毫升的吸管并用重悬血液。</li>
	</ol>
</li>
	<li>
DSS 中的血清斑点
	<ol>
<li>用吸管将一点血清 (50 µL) 转移到保护卡上。重复这一步, 有一个总的3–5血点。 注意: 要非常小心地将50µL 的血清注入每个部位。血清不应超出卡的虚线。</li>
		<li>让 DSS 在室温下过夜。把保护卡的盖子打开, 方便血液干燥。</li>
		<li>将卡在室温下存放在带有干燥剂的塑料袋中, 直到使用。如果存储时间超过14天, 请将卡存储在-20 摄氏度。</li>
	</ol>
</li>
</ol>4. 从 DSS 开始的 ELISA 检测
<ol><li>
洗脱蛋白来自 DSS。
	<ol>
<li>如有必要, 解冻每个示例的 DSS。将 DSS 切成小块 (直径约1毫米)。把这些碎片放入一个1.5 毫升管与400µL PBS。</li>
		<li>在4摄氏度一夜之间摇动样品。 注: 在低强度下摇动。在震动时, PBS 不应将管子的盖子弄湿。</li>
	</ol>
</li>
	<li>根据 ELISA 试剂盒的指示, 在适当的稀释后, 使用蛋白洗脱液作为血清标本进行分泌蛋白/生长因子分析。 注: 本程序适用于 DBP 检测9。对于其他 ELISA 化验, 可能需要一个优化步骤。</li>
</ol>

<ol>
	<li>Bertagnolio, S., 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=HIV-1+drug+resistance+surveillance+using+dried+whole+blood+spots.">HIV-1 drug resistance surveillance using dried whole blood spots.</a> Antivir Ther. 12 (1), 107-113 (2007).</li><li>Mei, J. V., Alexander, J. R., Adam, B. W., Hannon, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+filter+paper+for+the+collection+and+analysis+of+human+whole+blood+specimens.">Use of filter paper for the collection and analysis of human whole blood specimens.</a> J Nutr. 131 (5), 1631S-1636S (2001).</li><li>Garrido, C., 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=Subtype+variability,+virological+response+and+drug+resistance+assessed+on+dried+blood+spots+collected+from+HIV+patients+on+antiretroviral+therapy+in+Angola.">Subtype variability, virological response and drug resistance assessed on dried blood spots collected from HIV patients on antiretroviral therapy in Angola.</a> J Antimicrob Chemoth. 61 (3), 694-698 (2008).</li><li>Steegen, K., 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=Feasibility+of+detecting+human+immunodeficiency+virus+type+1+drug+resistance+in+DNA+extracted+from+whole+blood+or+dried+blood+spots.">Feasibility of detecting human immunodeficiency virus type 1 drug resistance in DNA extracted from whole blood or dried blood spots.</a> J Clin Microbiol. 45 (10), 3342-3351 (2007).</li><li>Costenaro, P., 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=Viral+load+detection+using+dried+blood+spots+in+a+cohort+of+HIV-1-infected+children+in+Uganda:+correlations+with+clinical+and+immunological+criteria+for+treatment+failure.">Viral load detection using dried blood spots in a cohort of HIV-1-infected children in Uganda: correlations with clinical and immunological criteria for treatment failure.</a> J Clin Microbiol. 52 (7), 2665-2667 (2014).</li><li>Gong, Z. H., Tian, G. L., Huang, Q. W., Wang, Y. M., Xu, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+glutathione+and+glutathione+disulfide+in+the+blood+of+glucose-6-phosphate+dehydrogenase-deficient+newborns.">Reduced glutathione and glutathione disulfide in the blood of glucose-6-phosphate dehydrogenase-deficient newborns.</a> BMC Pediatr. 20 (17), 172 (2017).</li><li>Lukacs, Z., Barr, M., Hamilton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practice+in+the+measurement+and+interpretation+of+lysosomal+acid+lipase+in+dried+blood+spots+using+the+inhibitor+Lalistat+2.">Best practice in the measurement and interpretation of lysosomal acid lipase in dried blood spots using the inhibitor Lalistat 2.</a> Clin Chim Acta. 471, 201-205 (2017).</li><li>Skogstrand, P. T., Bent, N. P., Schendel, D. E., S&#248;rensen, L. C., Hougaard, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+25+inflammatory+markers+and+neurotrophins+in+neonatal+dried+blood+spots+by+immunoassay+with+xMAP+technology.">Simultaneous measurement of 25 inflammatory markers and neurotrophins in neonatal dried blood spots by immunoassay with xMAP technology.</a> Clin Chem. 51 (10), 1854-1866 (2005).</li><li>Amadori, 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=Vitamin+D+receptor+polymorphisms+or+serum+levels+as+key+drivers+of+breast+cancer+development?+The+question+of+the+vitamin+D+pathway.">Vitamin D receptor polymorphisms or serum levels as key drivers of breast cancer development? The question of the vitamin D pathway.</a> Oncotarget. 8 (8), 13142-13156 (2017).</li><li>Gupta, B. P., Jayasuryan, N., Jameel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+hepatitis+B+virus+from+dried+blood+spots+by+polymerase+chain+reaction+amplification.">Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification.</a> J Clin Microbiol. 30 (8), 1913-1916 (1992).</li><li>Colson, K. E., Potter, A., Conde-Glez, C., Hernandez, B., R&#237;osZertuche, D., Z&#250;&#241;iga-Brenes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+commercial+ELISA+for+the+detection+of+measles-specific+immunoglobulin+G+(IgG)+in+dried+blood+spots+collected+from+children+living+in+low-resource+settings.">Use of a commercial ELISA for the detection of measles-specific immunoglobulin G (IgG) in dried blood spots collected from children living in low-resource settings.</a> J Med Virol. 87 (9), 1491-1499 (2015).</li><li>Drabe, C. H., Blauenfeldt, T., Ruhwald, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA-based+assay+for+IP-10+detection+from+filter+paper+samples.">ELISA-based assay for IP-10 detection from filter paper samples.</a> Methods Mol Biol. 1172, 27-37 (2014).</li><li>St Julien, K. R., 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=High+quality+genome-wide+genotyping+from+archived+dried+blood+spots+without+DNA+amplification.">High quality genome-wide genotyping from archived dried blood spots without DNA amplification.</a> PLoS One. 8 (5), e64710 (2013).</li><li>Shaner, R. L., Schulze, N. D., Seymour, C., Hamelin, E. I., Thomas, J. D., Johnson, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+fentanyl+analogs+in+dried+blood+spots+by+flow-through+desorption+coupled+to+online+solid+phase+extraction+tandem+mass+spectrometry.">Quantitation of fentanyl analogs in dried blood spots by flow-through desorption coupled to online solid phase extraction tandem mass spectrometry.</a> Anal Methods. 9, 3876-3883 (2017).</li></ol>

作者没有披露的利益冲突。

本议定书研究当实验室没有必要的工作人员或基础设施来正确处理血液样本时, 在滤纸上储存血液和血清的潜力。特别是, 在标准管或手指刺中收集的全血或血清可以用这种方法储存, 并且没有必要在血液收集后立即冷冻-20 摄氏度或-80 摄氏度的样品。星展/DSS 可以保持室温14天, 不改变血液/血清完整性。储存的一个关键问题是水分的存在, 影响生物材料的性能超过温度。使用干燥剂的塑料袋可以避?...

研究癌症生物标志物的主要目的是确定新的生物学参数, 用于诊断、预测患者预后, 以及确定患者是否会对特定治疗作出反应。这个研究领域是发现创新癌症治疗的基础, 并在量身定做的治疗中发挥关键作用。
在生物标志物鉴定和验证的每一个步骤中执行的程序必须是可靠和可重现的。翻译研究成功的基石是正确储存血液和血清等生物样本。这是获得高质量生物材料的第一步, 可用于进行分子生物学实验或蛋白质化验。
多中心的研究往往需要招募足够的病人, 以获得可靠的数据。并非所有的研究所都能将样品储存在-80 摄氏度, 或者将样品送到其他国际中心的干冰中。使用滤纸收集血液是一种简单的方法来储存血液和血清, 不需要立即冻结样本1,2。一滴血或血清可以被发现在纸上, 留到干燥过夜, 然后储存14天的室温1,2。这使得研究者们有时间把样品送到其他实验室。利用干血点 (DBS) 和干血清点 (DSS) 可以简化发达国家和发展中国家研究所之间的合作。
由于它的易用性, DBS 取样被广泛应用于几种血清学或遗传下游分析的化验。例如, 在过去, 星展集团经常被用于发展中国家的艾滋病毒筛查1、2、3、4、5。这种存储方法的另一个优点是可以从手指刺中收集血液样本, 从而使其用于新生儿筛查试验6、7、8。方便的样品处理和运输是 DBS 的进一步优势, 特别是在没有实验室设备的偏远地区收集的样品。在以前的出版物中, 我们使用 DBS 和 DSS 测试维生素 d 和维生素 d 结合蛋白 (DBP) 在一系列的白种人和非洲患者9。我们的非洲同事无法获得干冰。为了比较生物标记物对两个族群之间维生素 D 通路的差异, 我们进一步改进了在标准条件下和滤纸上储存的匹配样品的使用方法。在优化了星展/DSS 程序后, 我们能够分析两个患者血清中的 DBP 和维生素 D。我们还评估了一些单核苷酸多态性 (SNPs) 后, 从全血 DNA 提取的白种人和星展集团的非洲9。本议定书允许高质量的血液样本储存在室温下, 而不影响不同类型的下游分析, 从分子生物学到 ELISA 化验。建议用于管理多中心研究中的生物材料或没有标准贮存条件设施的中心。下面的协议代表了这些优化过程的顶点。

<table>
	<tbody>
		<tr>
			<td>Whatman protein saver cards 903 Protein saver card, 100/pk&#8206;</td>
			<td>Sigma</td>
			<td>Z761575</td>
			<td>Useful to store samples at room temperature for downstream analyses</td>
		</tr>
		<tr>
			<td>Falcon Serological Pipettes, 5 mL</td>
			<td>Stem cell</td>
			<td>#38003</td>
			<td></td>
		</tr>
		<tr>
			<td>50-200 &#181;L tips</td>
			<td>Star-Lab</td>
			<td>S1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>Eppendorf</td>
			<td>4036-3204</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA microkit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td>This is a DNA extraction kit designed to isolate small quantities of DNA&#160;&#160;</td>
		</tr>
		<tr>
			<td>Buffer ATL (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Lysis Buffer 1 in the text</td>
		</tr>
		<tr>
			<td>Buffer AL (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Lysis Buffer 2 in the text</td>
		</tr>
		<tr>
			<td>QIAamp mini elute column</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as column in the text</td>
		</tr>
		<tr>
			<td>AW1&#160; (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Wash Buffer 1 IN THE TEXT</td>
		</tr>
		<tr>
			<td>AW2&#160; (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Wash Buffer 2 in the text</td>
		</tr>
		<tr>
			<td>Human Vitamin D BP Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>DVDBP0</td>
			<td></td>
		</tr>
	</tbody>
</table>

dried blood and serum spots as a useful tool for sample storage to evaluate cancer biomarkers

血液样本质量是确保准确的下游分析, 如实时 PCR 或 ELISA 的关键。正确贮存生物材料是实现可重现性和可靠结果的出发点。所有样品的处理方式应与采集血液的方法相同。根据要执行的分析, 整个血液和血清样本应储存在-20 摄氏度或-80 °c, 直到使用。血液/血清标本也应 aliquoted, 以避免多种冻融。另一个重要的问题是从一个实验室装运到另一间化验室的样品条件。如果干冰不可用或装运时间超过几天, 则需要其他方法。一种选择是使用滤纸收集血液。在这里, 我们提出了血液和血清标本收集的方法, 利用干血点 (DBS) 和干血清点 (DSS)。我们开发了从 DBS 中提取 DNA 的程序, 用于对某些单核苷酸多态性 (SNPs) 进行实时 PCR 的下游评价。我们还优化了从 DSS 蛋白质洗脱开始的 ELISA 检测。该方法可与其他酶联免疫测定法或评估蛋白质的程序一起使用。

我们利用星展银行和 DSS 的程序, 在室温下储存血液和血清, 而不影响生物材料的质量。图 1显示了一个没有血液和血液收集后的蛋白卡保护程序的例子。为了确认滤纸的贮存和洗脱血液的过程不影响样品质量, 我们对标准贮存方法进行了比较。我们提取的 DNA 来自3匹配样本的全血收集的2毫升管或存储为 DBS, 如报告的协议部分。采用实时 PCR 技术对五个...

Watch this Scientific Journal Video about Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers at JoVE.com

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

该协议描述了一种简单有效的方法, 用于存储外周血和血清/血浆, 用于下游分析, 如单核苷酸多态性 (SNP) 评价和 ELISA 检测。

干血和血清斑点作为样品贮存的有用工具, 用于评估肿瘤生物标志物

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

dried-blood-serum-spots-as-useful-tool-for-sample-storage-to-evaluate

Research

JoVE Journal

Cancer Research

10.4K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy. 该协议描述了一种简单有效的方法, 用于存储外周血和血清/血浆, 用于下游分析, 如单核苷酸多态性 (SNP) 评价和 ELISA 检测。

视频: Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

科研

癌症研究

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

酵母作为开发功能分析研究人类P53的机箱

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

使用转录组学分析绘制无序致癌转录因子的结构-功能关系

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

使用 E1A 微型工具研究 mRNA 拼接更改

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...