<p class="jove_content"&gt; RN部分资金由居里夫人欧洲重新融入社会资助（ERG）从欧盟第七框架计划，项目编号268324。</p&gt;

1。从人体血液中的中性粒细胞密度离心分离<ol><li>抽取5毫升管静脉血肝素。 </li><li>准备双层聚蔗糖梯度（4梯度为5管血）如下：倒入12毫升的密度1.119克/毫升在50毫升管中的聚蔗糖溶液中，并小心地层10毫升的密度1.077克/毫升的聚蔗糖溶液的顶部。 </li><li>用等体积的PBS中，稀释血液。 </li><li>层仔细双层聚蔗糖梯度稀释血液; 20-25毫升，每梯度。 </li><li>以396×g离心20分钟，一个吊桶转子，22°C无制动。 </li><li>准备冷RPMI含有0.05％的人血清白蛋白（RPMI-HSA）。还要准备9毫升无菌去离子H 2 O预冷的RPMI和H 2 O冰。 </li><li>吸出含有等离子体（黄色）和PBMC的最佳聚蔗糖层和第二层的聚蔗糖（白色），使用用真空泵（把一个无菌移液管尖移液管）。 </li><li>中性粒细胞收集在50毫升的试管，用一个小塑料吸管（1管，中性粒细胞每2梯度分数），并把它们放在冰。 </li><li>加入冷的RPMI-HSA的总体积为50毫升，离心在4℃下以249×g的10分钟</li><li>脱掉上清液用真空泵的使用和涡流颗粒轻轻（红细胞和中性粒细胞）。 </li><li>加入9毫升无菌去离子H 2 O和启动定时器。 30秒后完全停止超渗透压冲击，加入1 ml 10倍浓缩的PBS。注：30秒是非常关键的。 </li><li>加入冷的RPMI-HSA的总体积为50毫升，离心在4℃下以249×g的10分钟</li><li>脱掉上清液与一真空泵的使用和收集的中性粒细胞的颗粒在1管与定义的体积（1-2毫升）的RPMI-HSA。 </li><li>确定的细胞量的浓度调整到1.10 7个细胞/毫升。根据供体的，孤立的中性粒细胞的产率将是9:13连接5×10 6 3×10 7的中性粒细胞从每个9毫升管血</li></ol> 2。流式细胞仪检测方法的评估钙动员人力中性粒细胞<ol><li>负载细胞（5×10 6细胞/ ml）与2μM氟-3 - AM在RPMI-HSA和孵育20分钟，在室温下缓慢地摆动，例如，一个使用摆动平台的振动筛，避光。 </li><li>在此期间准备的刺激终浓度在10倍系列稀释（ 例如 3倍）。 PSMα3在这个协议中使用，但任何作用于嗜中性粒细胞的G蛋白偶联受体的刺激是合适的。 </li><li>准备10倍浓缩RPMI-HSA受体抑制剂。当抑制剂在室温下10分钟，用等体积的抑制剂作用于预孵育25μl的刺激的刺激（ 例如，高密度脂蛋白年PSMα3）。 </li><li>洗涤细胞，加入10ml的RPMI-HSA，并在室温下离心249×g离心。悬浮细胞，以5×10 6细胞/ ml。 </li><li>在实验前，稀释细胞以2×10 6个细胞/毫升的RPMI-HSA和每FACS管中加入200μl的细胞。稀释后的中性粒细胞是很脆弱的。太长时间保持他们在此浓度下，会导致自动激活，也同样适用于剧烈摇晃或吹打。 </li><li>连接管的流式细胞仪，等待3秒，并开始收购。经过一个固定的时间周期（ 例如 8秒），取下管，迅速 ​​加入50微升样品的刺激。立即重新连接到样品架管，并继续收购。 </li><li>开始的最低和最高浓度的刺激。 RPMI-HSA每次运行后定期清洗流式细胞仪针。 </li><li>流式细胞仪分析软件分析数据。比较平均的荧光信号的刺激，然后加入另外的刺激后，使用适当的阳性和阴性对照，计算交流的tivati​​on强度测量每个稀释度。 </li></ol>或者抑制剂时，作用于受体与抑制剂的细胞预孵育。 3。中性粒细胞吞噬细菌GFP基因的表达后荧光显微镜分析<ol><li>成长S.金黄色葡萄球菌菌株记者晚上在利益结构肉汤（用抗生素时，须维持记者质粒）。在这种情况下，应变MW2含有GFP报告一个PSMα的构造是用来10，用5ml的LB培养基中生长在一个50毫升的塑料管。 </li><li>要删除所有O / N表达GFP，淡化文化外径660 0.01增长到外径660 0.1。 Redilute这种文化1:30和监控增长，直到文化达到0.1外径660。离心收集细菌，并在DPBS洗一次。重悬在1:10原体积的取得的OD 660为1.0或roughly 5.10 8 CFU /毫升。 </li><li>与新鲜分离的中性粒细胞在RPMI-HSA（比例10:1）（1.10 6 /毫升）1.5毫升的微管中的细菌（1.10 7个/ ml）混合，并添加至终浓度为10％合并的人血清。摇上摇10分钟的平台，在37°C至刺激的吞噬功能。装有细菌5.10 5中性粒细胞/ ml和吸取250微升入井8腔盖玻片稀释的中性粒细胞。 </li><li>中性粒细胞与细菌在显微镜图片。 40X/0.85 NA物镜具有一个倒置显微镜配备工作良好，应包裹在黑暗的环境下腔室，保持环境稳定在37℃。一些使用相机的预设位置，每隔5-10分钟亮场和GFP的通道，按照GFP的生产时间获取图像。 </li></ol>

<ol>
	<li>Rigby, K. M., DeLeo, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+innate+host+defense+against+Staphylococcus+aureus+infections.">Neutrophils in innate host defense against Staphylococcus aureus infections.</a> Semin. Immunopathol. 34, 237-259 (2012).</li><li>Voyich, J. M., 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=Insights+into+Mechanisms+Used+by+Staphylococcus+aureus+to+Avoid+Destruction+by+Human+Neutrophils.">Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils.</a> The Journal of Immunology. 175, 3907-3919 (2005).</li><li>Kobayashi, S. 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=Rapid+neutrophil+destruction+following+phagocytosis+of+Staphylococcus+aureus.">Rapid neutrophil destruction following phagocytosis of Staphylococcus aureus.</a> J. Innate Immun. 2, 560-575 (2010).</li><li>DeLeo, F. R., Otto, M., Kreiswirth, B. N., Chambers, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-associated+meticillin-resistant+Staphylococcus+aureus.">Community-associated meticillin-resistant Staphylococcus aureus.</a> The Lancet. 375, 1557 (2010).</li><li>David, M. Z., Daum, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-Associated+Methicillin-Resistant+Staphylococcus+aureus:+Epidemiology+and+Clinical+Consequences+of+an+Emerging+Epidemic.">Community-Associated Methicillin-Resistant Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging Epidemic.</a> Clinical Microbiology Reviews. 23, 616-687 (2010).</li><li>Kretschmer, 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=Human+Formyl+Peptide+Receptor+2+Senses+Highly+Pathogenic+Staphylococcus+aureus.">Human Formyl Peptide Receptor 2 Senses Highly Pathogenic Staphylococcus aureus.</a> Cell Host &amp; Microbe. 7, 463 (2010).</li><li>Prat, 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=A+homolog+of+formyl+peptide+receptor-like+1+(FPRL1)+inhibitor+from+Staphylococcus+aureus+(FPRL1+inhibitory+protein)+that+inhibits+FPRL1+and+FPR.">A homolog of formyl peptide receptor-like 1 (FPRL1) inhibitor from Staphylococcus aureus (FPRL1 inhibitory protein) that inhibits FPRL1 and FPR.</a> J. Immunol. 183, 6569-6578 (2009).</li><li>Prat, C., Bestebroer, J., de Haas, C. J. C., van Strijp, J. A. G., van Kessel, K. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Staphylococcal+Anti-Inflammatory+Protein+That+Antagonizes+the+Formyl+Peptide+Receptor-Like+1.">A New Staphylococcal Anti-Inflammatory Protein That Antagonizes the Formyl Peptide Receptor-Like 1.</a> The Journal of Immunology. 177, 8017-8026 (2006).</li><li>de Haas, C. J., 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=Chemotaxis+inhibitory+protein+of+Staphylococcus+aureus,+a+bacterial+antiinflammatory+agent.">Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent.</a> The Journal of Experimental Medicine. 199, 687-695 (2004).</li><li>Surewaard, B. G. J., 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=Inactivation+of+Staphylococcal+Phenol+Soluble+Modulins+by+Serum+Lipoprotein+Particles.">Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles.</a> PLoS Pathog. 8, e1002606 (2012).</li><li>Kubica, M., 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=A+Potential+New+Pathway+for+Staphylococcus+aureus+Dissemination:+The+Silent+Survival+of+S.+aureus+Phagocytosed+by+Human+Monocyte-Derived+Macrophages.">A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages.</a> PLoS ONE. 3, e1409 (2008).</li><li>Surewaard, B. G. J., 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=Staphylococcal+alpha-phenol+soluble+modulins+contribute+to+neutrophil+lysis+after+phagocytosis.">Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis.</a> Cellular Microbiology. , (2013).</li><li>Queck, S. Y., 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=RNAIII-Independent+Target+Gene+Control+by+the+agr+Quorum-Sensing+System:+Insight+into+the+Evolution+of+Virulence+Regulation+in+Staphylococcus+aureus.">RNAIII-Independent Target Gene Control by the agr Quorum-Sensing System: Insight into the Evolution of Virulence Regulation in Staphylococcus aureus.</a> Molecular Cell. 32, 150 (2008).</li><li>Baban, C. 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=Bioluminescent+Bacterial+Imaging+In+Vivo.">Bioluminescent Bacterial Imaging In Vivo.</a> J. Vis. Exp. (69), e4318 (2012).</li></ol>

作者宣称，他们有没有竞争经济利益。

在这里描述的方法中，一些步骤是非常关键的。我们将突出这些在这里。 如果在中性粒细胞通过密度梯度离心的分离，重要的是离心步骤期间或之后，为了不妨碍层。当使用塑料吸管吸嗜中性粒细胞，确保不要挤气球，而在细胞层，喷射液体会扰乱层。另外，目视检查后中性粒细胞颗粒渗透冲击和离心步骤。如果沉淀仍然是红色的红细胞裂解并不足够有效的，应再重复一次?...

中性粒细胞（中性粒细胞）是专业吞噬细胞发挥 ​​了关键作用，对金黄色葡萄球菌的先天免疫反应。主机和微生物之间不断的战斗已导致双方的军备竞赛。近日，社区相关（CA）株耐甲氧西林S.金黄色葡萄球菌 （MRSA）的出现，似乎是非常有效的规避中性粒细胞杀2,3。过量的苯酚，水溶性modulin（PSM）的生产的CA-MRSA一直伴随着较高的毒力4,5。人类嗜中性粒细胞能识别这些的PSM通过的FPR2导致活化的G蛋白偶联受体6。最早的事件之一是动员细胞内储存的钙（Ca 2 +的）。 Ca 2 +的作用作为第二信使的效应子功能的各种中性粒细胞的脱颗粒和吞噬作用7。因此的Ca 2 +是一个非常敏感的指标的PSM激活中性粒细胞功能的能力。的PSM对中性粒细胞的影响进行研究，新鲜的嗜中性粒细胞分离和钙动员后，荧光染料装入。流式细胞仪在实验中，这种快速的动员可以衡量的。使用这种方法，可以研究对中性粒细胞的毒性和其它组件的直接影响，并确定这些活性的最低浓度。对于我们来说，这是一个非常有用的工具来研究许多蛋白质产生的效果由S.金黄色葡萄球菌参与免疫逃避，如的FPR2抑制蛋白（FLIPR）8，FLIPR像7，抑制趋化蛋白的金黄色葡萄球菌 （筹）9。所有这些蛋白质已被证明能够抑​​制嗜中性粒细胞中钙的动员，通过结合受体识别激动剂。 最近，我们的组描述的PSM功能抑制血清脂蛋白10 &lt;/ SUP&gt;。这些脂蛋白是大量存在于血液和人体组织，表明的PSM主要在细胞内环境中发挥其功能。钙动员检测的可用性允许激活中性粒细胞的PSM所表示的相当大的抑制血清中的浓度非常低，我们能够精确地测量血清脂蛋白的影响。 的PSM功能抑制血清中，我们假设，作为细胞内毒素的PSM是一个重要的功能。因此，我们试图确定后吞噬作用的PSM。要调查的PSM间隙的表达，我们已经建立了记者操纵psmαGFP融合的推动者。当这些记者菌株的的中性粒细胞吞噬金黄色葡萄球菌 ，观察到诱导表达用荧光显微镜10。显然，这样的构造共鸣装置允许大量的在S基因的表达研究后吞噬金黄色葡萄球菌或其他病原体。由于为S。间利基尚存在金黄色葡萄球菌是非常重要的是要克服先天免疫系统的11 10 12，在这个利基激活的基因的作用研究，以了解其毒力是高度相关的。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Fluo-3, AM</td>
			<td>Molecular Probes / Life Technologies</td>
			<td>F-1241</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-5442-03</td>
			<td>density 1.077 g/ml</td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>11191</td>
			<td>density 1.119 g/ml</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco, Life Technologies</td>
			<td>52400-025</td>
			<td>contains 25 mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>Leica TCS SP5 microscope</td>
			<td>Leica Microsystems, The Netherlands</td>
			<td>TCS SP5</td>
			<td>objective: HCX PL APO 40X/0.85</td>
		</tr>
		<tr>
			<td>FACSCalibur</td>
			<td>BD Biosciences</td>
			<td>FACSCalibur</td>
			<td>Very important that the tube can be removed and replaced during the measurement process</td>
		</tr>
	</tbody>
</table>

studying interactions of staphylococcus aureus with neutrophils by flow cytometry and time lapse microscopy

我们提出的方法来研究苯酚的可溶性modulins（PSM）的和其他毒素的金黄色葡萄球菌对中性粒细胞产生和分泌的效果。为了研究中性粒细胞的PSM隔离新鲜的中性粒细胞，采用密度梯度离心的效果。这些中性粒细胞钙动员后，荧光染料装入。激活中性粒细胞的PSM启动一个细胞内游离钙离子浓度的增加迅速而短暂的。在流式细胞仪实验，通过监视一个预载的荧光染料，反应的游离Ca 2 +浓度的增加，可以测量这种快速动员。使用这种方法，我们可以判断的的PSM浓度要激活的嗜中性粒细胞，并测量具体和一般的嗜中性粒细胞活化的抑制剂的影响。 要探讨的PSM中的表达，在细胞内的空间，瓦特我们构建操纵PSMαGFP融合的推动者记者。当这些记者菌株的黄色葡萄球菌的中性粒细胞吞噬，诱导表达可以用荧光显微镜观察。

流式细胞仪检测评估钙动员人体中性粒细胞孵化产生的合成PSMα3的钙离子流的增加在FL-1中的信号，这是显示所测得的快速激活的嗜中性粒细胞的浓度系列。预孵育合成PSMα3与0.01％，0.1％或1％人血清显着抑制的能力引起的钙通量（ 图1）。 中性粒细胞吞噬后在细菌中表达的GFP荧光显微镜分析吞噬细菌含有PSMαGFP记者10日</sup...

Watch this Scientific Journal Video about Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy at JoVE.com

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

我们提出的方法来研究的PSM和其他分泌的毒素的效果<em&gt;金黄色葡萄球菌</em&gt;中性粒细胞，使用流式细胞仪和荧光显微镜。

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

studying-interactions-staphylococcus-aureus-with-neutrophils-flow

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JoVE Journal

Immunology and Infection

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

13.0K 次观看.  University Medical Center Utrecht. 我们提出的方法来研究的PSM和其他分泌的毒素的效果金黄色葡萄球菌中性粒细胞，使用流式细胞仪和荧光显微镜。

视频: Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

科研

免疫与感染

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

皮下感染耐甲氧西林金黄色葡萄球菌（MRSA）的

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

金黄色葡萄球菌生长使用人血红蛋白作为铁源

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

方法基因水平转移的研究<em&gt;金黄色葡萄球菌</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...