我们感谢我们的技术助理: U. Bröder 和 h. Shkarlet, 他在实验过程中帮助我们。这项工作部分是由欧盟赠款 (UniVax, 601738 号合同和 TRANSVAC2, 730964号合同) 和亥姆霍兹协会赠款 (海-IDR) 资助的。资金来源不影响研究设计, 生成手稿或决定提交出版。

所有在这项研究中使用的老鼠都是从 C57BL/6 的背景。所有的动物都被保存在无病原体条件下。所有实验都是根据德国动物保护法 (TierSchG BGBl) 的规范进行的。我是 1105;25.05.1998), 并获下萨克森动物实验伦理学委员会和国家办事处 (下萨克森国家消费者保护和食品安全办公室) 批准, 许可证号为 33.4-42502-04-13/1281 和162280。
1. CFSE 细胞染色及移植
注意: OT I 小鼠是 transgenically 生成的动物, 表达 T 细胞受体 (TCR) 与固定α和β链, 共同承认的免疫优势肽的卵子, SIINFEKL10,11。结果, 这些小鼠有相当大数量的 SIINFEKL 特异性 CD8+ T 细胞 (97%)12 , 当比较正常或卵免疫小鼠 (≤ 1%)13。
<ol><li>Thy1a (Thy1.1 ) 等位基因表达 T 淋巴细胞特异性的 CD8 t 细胞的分离<ol>
<li>弄死6-9 周大鼠, 由 CO2吸入, 其次为颈椎脱位14。解剖脾脏和主要淋巴结 (腹股沟, 腋窝和颈部对只) 通过切割皮肤与外科剪刀, 从身体分离的皮肤与外科钳和镊子的帮助下, 并将其放置在培养皿 (60 x 15 毫米) 每个包含一个100µm 孔网杯, 用于组织研磨和细胞释放。</li>
		<li>在3-5 毫升完整的 RPMI 培养基 (RPMI 1640, 10% 伏/v FCS, 100 U/毫升青霉素, 50 µg/毫升链霉素) 保持在冰上的培养皿 (每一个网杯)。</li>
		<li>在注射器柱塞或相似的不育仪器的帮助下捣碎器官 (不需要事先切开) 并且收集导致的单细胞悬浮在15毫升离心机管子。</li>
		<li>离心机细胞悬浮在 300 x g 10 分钟在4°c。放弃上清。将10毫升的冷 PBS 中的细胞通过离心和溶解, 在氯化铵缓冲液1毫升/脾中重新悬浮颗粒 (ACK 缓冲器, 商用), 将红细胞从脾脏中冲洗出来。在冰上孵化细胞1.5 分钟, 然后用离心法 (如前所做) 用10毫升的冷 PBS 冲洗细胞。</li>
		<li>在1毫升的 PBS (pH 7.2) 中, 用5% 胎牛血清进行磁隔离, 将颗粒重新悬浮在同一管中。</li>
	</ol>
</li>
	<li>要进行磁隔离, 请根据制造商指令或已发布的协议15, 使用磁性隔离套件进行 CD8+ T 细胞的阴性选择。</li>
	<li>要执行 CFSE 染色, 首先计算使用自动单元计数器 (粒子计数器) 从 OT I 小鼠获得的 CD8+ T 细胞数。着色 1-5 x 107细胞/毫升在1-5 毫升的容量与5µM CFSE 在 PBS 为7分钟在37°c, 保护免受光在15毫升猎鹰管。</li>
	<li>将相同体积 (1:1) 的胎牛血清添加到细胞中, CFSE 染色, 并在37摄氏度的保护下, 将其孵化7分钟。用10毫升 PBS 清洗细胞两次。</li>
	<li>使用自动单元格计数器计数单元格。将细胞数设置为 3-5 x 107细胞/毫升的 PBS, 以注射 3-5 x 106细胞/小鼠在100µL 静脉 (静脉注射) 通过尾静脉。</li>
	<li>进行尾静脉注射, 将小鼠固定在适当的抑制剂。加热后区和尾部的小鼠被注射使用红光灯, 放置在20至25厘米以外的小鼠1-3 分钟, 以允许尾部静脉舒张。 注意: 这有助于缓解静脉检测和细胞悬浮的管理。</li>
	<li>确保 (与鼠标和灯之间的手), 它是不太热的老鼠, 当尾巴静脉清楚可见, 进行注射。用25口径针16的1mL 注射器将细胞注入侧或背尾静脉。</li>
</ol>2. 免疫接种 (游离卵子 +/-佐剂)
<ol><li>将小鼠与 OT I 细胞 (步骤 1.7,图 1) 移植到麻醉室中, 用麻醉机14在氧气中管理异氟醚。</li>
	<li>当老鼠在麻醉下完全睡着的时候, 把它从房间里拿出来, 在臀部 superficialis (侧下背部) 的地方剃掉小鼠的皮毛, 用电动理发机, 以执行干净的注射。对应用领域有很好的看法。</li>
	<li>注射50µL 的疫苗, 在被剃光区域使用25口径针。</li>
</ol>3. 流式细胞术分析中淋巴细胞和染色的分离
<ol><li>淋巴细胞与脾的分离<ol>
<li>弄死联合2吸入免疫接种小鼠, 其次为颈椎脱位。</li>
		<li>提取引流和远端淋巴结和脾脏, 并将其放置在单独的培养皿中, 含有100µm 孔网杯用于组织研磨和细胞释放。按照步骤1.1.2 通过1.1.3。</li>
		<li>离心后醒酒上清液, 并在相同体积的残余 PBS 中重新悬浮细胞颗粒 (约100µL)。</li>
	</ol>
</li>
	<li>流式细胞术分析染色<ol>
<li>在一个15毫升离心管准备一个主混合含有染色抗体的浓度在表 1中, 从而产生2x 浓缩染色混合。准备足够数量的主混合, 每样有100µL。混合细胞和主混合1:1 和孵化它在 4°c 30 分钟。 注: 每个样品的最终染色量应为200µL (100 µL 细胞颗粒 +100 µL 抗体主混合)。</li>
		<li>1.1.3 中描述的离心两次清洗细胞, 增加10毫升的 PBS, 两次。</li>
		<li>在 0.5-1 毫升的 PBS 中重新悬浮染色细胞, 用于采集和转移悬浮液, 用于流式细胞仪管。总是把样品放在冰上, 以免被光线所保护。</li>
	</ol>
</li>
</ol>4. 流式细胞仪
<ol><li>始终使用70-100 µm 过滤器预先筛选单元格样本。</li>
	<li>为表 1 (珠子或细胞) 中详细的显影准备单一染色补偿控制。 注: 应在细胞仪或分析软件17、18、19中进行补偿, 并应用于所有样品。</li>
	<li>按照图 2中描述的浇口策略进行。简要地, 门人口在正向散射高度与正向散射区域 (图 2A), 并且再侧散布宽对侧散布区域 (SSA,图 2B) 为了排除双峰。</li>
	<li>通过绘制 BV 650 (自动荧光) 与 FITC (CFSE) 来区分来自高自动荧光细胞的真实 CFSE 染色细胞, 从图 2B中进行填充。包括用与 BV 650 共轭的抗体染色的珠子或细胞纳入补偿控制。这一剧情 (图 2C) 的 BV 650 vs FITC 是用来门 CFSE 染色细胞。</li>
	<li>从图 2C中的 Pe-Cy7 (Thy1.1-CD90.1) 与 APC (CD8) 进行比较, 以区分来自捐赠者和受体小鼠的细胞。 注: 从 OT I. 捐献小鼠获得的细胞为 Thy1.1+ (CD90.1), 而 C57BL/6、受体) 小鼠派生细胞为 Thy1.2+ (CD90.2) (图 2D)。一些实验室有 Thy1.2 背景下的小鼠和 C57BL/6 (Thy1.1)。在这种情况下, 你必须使用抗体对抗 Thy1.2 细胞门。</li>
	<li>通过在 450 (CD4) vs APC (CD8) (图 2E) 的栅极上绘制 Thy1.1+填充的 CD8+细胞。</li>
	<li>通过使用显示 CFSE (FITC, 530/15 通道-蓝色激光) 的 CD8+单元格的直方图显示, 在图 2E中显示填充门, 通过将强度从 102增至水平处, 将激增的人口包括在内不分割控制人口是 (强度 ~ 105, 取决于 CFSE 染色的功效和流量式细胞仪的电压设置) (图 2F)。</li>
	<li>制作一个额外的门 (不显示在图2中) 绘制 FITC 与 L/D 标记 (450/20 通道, 紫外线激光), 以评估死细胞平行的扩散评估。</li>
	<li>从流式细胞仪的样本中获取至少5000项补偿控制和10.000 事件 (门 E,图 2) 的事件。在低或中流运行细胞仪 (不超过20.000 事件/秒的流速)。</li>
</ol>

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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=Type+I+interferon+protects+antiviral+CD8++T+cells+from+NK+cell+cytotoxicity.">Type I interferon protects antiviral CD8+ T cells from NK cell cytotoxicity.</a> Immunity. 40, 949-960 (2014).</li><li>Volk, A., 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=Comparison+of+three+humanized+mouse+models+for+adoptive+T+cell+transfer.">Comparison of three humanized mouse models for adoptive T cell transfer.</a> The journal of gene medicine. 14, 540-548 (2012).</li><li>Safinia, N., 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=Humanized+Mice+as+Preclinical+Models+in+Transplantation.">Humanized Mice as Preclinical Models in Transplantation.</a> Methods Mol Biol. 1371, 177-196 (2016).</li><li>Grover, A., 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=Humanized+NOG+mice+as+a+model+for+tuberculosis+vaccine-induced+immunity:+a+comparative+analysis+with+the+mouse+and+guinea+pig+models+of+tuberculosis.">Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.</a> Immunology. 152, 150-162 (2017).</li></ol>

作者没有什么可透露的。

现代疫苗是理想的 composedof 纯化抗原和佐剂, 有可能添加一个交付系统, 如脂质体, 病毒样粒子, 纳米粒子或活载体。设计疫苗的一个关键方面是根据临床需要选择合适的佐剂。部分的范围可能涉及倾向于体液与细胞免疫应答 (或两者), 选举局部与系统性免疫应答 (或两者), 以及这种疫苗必须在目标人群中产生的记忆。辅助评价的一个关键方面是迅速确定其产生 CTL 的能力。本文提出了一种基于已知?...

细胞毒性 T 淋巴细胞 (CTL) 诱导疫苗是为抗击某些类型的癌症而开发的关键治疗措施1。CTL 对预防性疫苗对细胞内病原体2也很重要。此外, ctl 是少数的免疫防御机制之一的功能活跃的风险人群, 如新生儿3,4谁也依赖 CTL, 以抗击早期生命感染5。在这方面, 与不引起 CTL 反应的佐剂一起研制的呼吸道合胞病毒疫苗 (明矾) 导致6婴儿感染严重并发症导致的疫苗完全失败。免疫接种的这些负面影响可以通过 CD8 细胞反应 7逆转。我们以前已经证明, 一些干扰素基因 (蜇) 激动剂诱发的主要细胞因子 (I. 干扰素) 对这些佐剂8产生的 CTL 反应至关重要, 部分是通过测量在疫苗接种后的 T 细胞, 并将这些结果作为在延长接种计划9中观察到的 CTL 诱导能力的量度。用羧基琥珀酰亚胺酯 (CFSE)染料稀释法测定野生型 (CD8) C57BL/6 受体小鼠中的 OT I. T 细胞的增殖是一种对疫苗佐剂能力的稳健估计, 可产生SIINFEKL 的交叉启动 (卵清蛋白的免疫主导肽, 卵)。这种技术的变异被广泛用于评估 CD8 和 CD4 T 细胞的增殖. 例如, 它已被用于没有选定的细胞因子 (高鼠) 或测量疫苗的功效后, 抗原召回的动物。我们设计了一个简短的协议 (4 天的实验), 在 CFSE 染色的 CD8+ T 细胞被动转移后, 皮下 (南卡罗来纳州) 免疫由一剂量50µg 的无内毒素卵子辅以测试佐剂被管理(图 1)。疫苗接种后48小时随访结果提供了可靠的证据证明佐剂产生 CTL 反应的能力。通过这一策略, 可以评估免疫后引流淋巴结局部免疫反应的效力, 以及测量脾脏 (或远端淋巴结) CTL 活动的反应程度。

<table>
	<tbody>
		<tr>
			<td>BD LSR Fortessa Cell Analyzer</td>
			<td>BD</td>
			<td>Special Order</td>
			<td>Flow Cytometer</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Molecular Probes</td>
			<td>C34554</td>
			<td>Proliferation Dye</td>
		</tr>
		<tr>
			<td>MojoSort Mouse CD8 T Cell Isolation Kit</td>
			<td>Biolegend</td>
			<td>480007</td>
			<td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
			<td>Molecular Probes</td>
			<td>L23105</td>
			<td>Dead Cell Marker</td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-0900-82</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD8a Antibody</td>
			<td>BioLegend</td>
			<td>100712</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>BV421 Rat Anti-Mouse CD4</td>
			<td>BD</td>
			<td>740007</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Z2 coulter Particle count and Size Analyzer</td>
			<td>Beckman Coulter</td>
			<td>9914591DA</td>
			<td>Cell counter. Z2 Automated particle/cell counter</td>
		</tr>
		<tr>
			<td>EndoGrade Ovalbumin (10 mg)</td>
			<td>Hyglos(Germany)</td>
			<td>321000</td>
			<td>Ovalbumin endotoxin free tested.</td>
		</tr>
		<tr>
			<td>Cell Strainer 100&#181;m nylon</td>
			<td>Corning</td>
			<td>352360</td>
			<td>Cell strainer (100 &#181;m pore mesh cups).</td>
		</tr>
		<tr>
			<td>Sample Vials</td>
			<td>Beckman Coulter</td>
			<td>899366014</td>
			<td>Sample vials for Z2 automated counter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice (CD90.2)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Company is now Envigo</td>
		</tr>
		<tr>
			<td>OT-I (C57BL/6 background, CD90.1)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Inbreed at our animal facility. Company from where adquired is now Envigo</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fischer (Corning)</td>
			<td>14-959-5</td>
			<td>Corning Falcon Round-Bottom Polystyrene Tubes</td>
		</tr>
		<tr>
			<td>Falcon 15 mL tubes</td>
			<td>Fischer (Corning)</td>
			<td>05-527-90</td>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>PBS (500 mL)</td>
			<td>Fischer (Gibco)</td>
			<td>20-012-027</td>
			<td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td>
		</tr>
		<tr>
			<td>Red lamp (heating lamp)</td>
			<td>Dirk Rossmann GmbH (Germany)</td>
			<td>405096</td>
			<td>Heating infrred lamp (100 wats)</td>
		</tr>
		<tr>
			<td>IsoFlo (Isoflurane)</td>
			<td>Abbott Laboratories (USA)</td>
			<td>5260.04-05.</td>
			<td>Isoflurane anesthesic (250 mL flask).</td>
		</tr>
		<tr>
			<td>Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber</td>
			<td>Parkland Scientific</td>
			<td>V3000PK</td>
			<td>Isoflurane anesthesia machine.</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>11-875-093</td>
			<td>Base medium with Glutamine (500 mL)</td>
		</tr>
		<tr>
			<td>Pen-Strept antibiotic solution (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>15-140-148</td>
			<td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>10082147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer (100 ml)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>A1049201</td>
			<td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td>
		</tr>
		<tr>
			<td>Plastic Petri Dishes</td>
			<td>Nunc (distributed by ThermoFischer)</td>
			<td>150340</td>
			<td>60 x 15mm Plastic Petri Dish, Non-treated</td>
		</tr>
		<tr>
			<td>Cell Clump Filter</td>
			<td>CellTrics (Sysmex)</td>
			<td>04-004-2317</td>
			<td>CellTrics&#174; 50 &#956;m, sterile</td>
		</tr>
	</tbody>
</table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

对现代分单元疫苗的评估表明, 中中和抗体的产生是重要的, 但不足以辅助选择。因此, 迫切需要具有体液和细胞免疫刺激能力的佐剂, 能够促进细胞毒 T 淋巴细胞 (CTL) 反应。因此, 对诱导交叉启动并随后增强 CTL 生成的佐剂候选者的忠实监测是疫苗开发的关键步骤。在这里, 我们提出一个方法的应用, 使用 SIINFEKL 特定 (OT) T 细胞监测模型抗原卵清蛋白 (卵)在体内的交叉呈现, 在不同的佐剂候选者在场。该方法是一种快速试验, 用于选择具有最佳交叉启动能力的佐剂。CD8+ T 细胞的增殖是交叉引发的最有价值的标志, 同时也被认为是佐剂诱导的交叉表达的相关性。这一特点可以评估在不同的免疫器官, 如淋巴结和脾脏。CTL 生成的程度也可以监测, 从而对局部 (引流淋巴结主要) 的性质或系统性反应 (远端淋巴结和/或脾脏) 有深入的了解。这项技术进一步允许对检测药物进行多次修改, 以抑制特定的交叉表达通路, 并提供了可能用于不同品系的常规和转基因小鼠。总之, 我们在这里提出的应用将是有用的在工业或学术界的疫苗实验室开发或修改疫苗研究和发展的化学佐剂。

为了测试使用不同佐剂 (ADJ1 和 ADJ2) 的治疗方法, 我们通过流式细胞仪测量过继转输转移的 CD8+ T 细胞的增殖来评估 CTL 的生成能力 (图 2)。为此, 我们以前从引流淋巴结和脾脏中分离出细胞 (表 1)。通过测量淋巴结和脾脏中 CD8+ T 细胞的增殖, 我们能够证实在引流淋巴结中 ADJ2 的 CTL 生成能力 (图 3) 与 A...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

我们在这里提出一个标准免疫技术 (CFSE 染色的应用), 旨在快速监测佐剂介导的细胞毒性 T 淋巴细胞 (CTL) 生成在体内.这种对 CTL 容量的快速估计有助于开发预防性疫苗, 防止细胞内病原体和治疗性癌症疫苗的发展。

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

rapid-vivo-assessment-adjuvant-s-cytotoxic-t-lymphocytes-generation

Research

JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K 次观看.  Helmholtz Centre for Infection Research. 我们在这里提出一个标准免疫技术 (CFSE 染色的应用), 旨在快速监测佐剂介导的细胞毒性 T 淋巴细胞 (CTL) 生成在体内.这种对 CTL 容量的快速估计有助于开发预防性疫苗, 防止细胞内病原体和治疗性癌症疫苗的发展。

视频: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

畸胎瘤代睾丸胶囊

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

科研

医学

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

快速点护理依诺肝素的抗凝血剂功效的测定全血中

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

光动力疗法治疗骨肉瘤细胞的细胞毒功效<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...