这项工作得到了1“的研究资助津贴”的克隆氏病的和结肠炎加拿大基金会的LMS，谁是1​​消化科/加拿大协会健康研究/克罗恩的小号和结肠炎加拿大新研究者奖基金会加拿大协会的支持。

1。使用含有氯磷酸二钠，脂质体的消耗巨噬细胞<ol><li>脂质体储存在4°C注射前两个小时，从冰箱中取出氯膦酸二钠含脂质体，PBS含脂质体，或无菌PBS（喷射控制），使他们能够适应新环境室温（18℃）。 </li><li>反转管含脂质体的8-10倍，以确保均匀分布，然后装入1 ml注射器200μL。将26号针头注射器顶端。 </li><li>用你的左手，浮渣鼠标，略低于持有足够的皮肤以固定其头部和四肢的耳朵。 </li><li>倾斜的鼠标，所以它的头是朝地面略等内脏器官从注射部位，这是位于腹部在较低的右象限移动。 </li><li>滚你的手掌之间的注射器和倒置的6倍，以散布均匀注射前脂质体的解决方案。 </li> &lt;李&gt;在鼠标腹部的右侧，在30-40度角插入针头。注入200μL脂质体溶液或PBS。 </li><li>引起的炎症模型中，像DSS诱导的结肠炎，注入小鼠实验性炎症的发病4天前，以确保在驻地巨噬细胞和每2天的消耗，消耗新的浸润巨噬细胞的协议。 </li><li>在实验结束后，随着计划中的实验分析，从网站的利益组织与多聚甲醛固定，确认巨噬细胞的免疫组织化学染色枯竭。 </li></ol> 2。推导极化巨噬细胞从骨髓穿刺<ol><li>从8至12周龄的小鼠骨髓骨髓来源的巨噬细胞的产量最高。安乐死的鼠标，把它在它的后面，并牵制它的爪子，尽可能伸展四肢。 </li><li>工作无菌胡汉清D，取出股骨和胫骨，尽量削减尽可能多的肌肉。 </li><li>装入装有26号针头5毫升注射器冲洗骨髓的骨头，用10％胎牛血清的IMDM培养。 </li><li>稀释骨髓抽吸至40毫升的IMDM培养与FCS的10％，地方75厘米2组织培养瓶细胞，并于37°C间，5％CO 2 4小时。这一步去除贴壁的造血祖细胞的间质细胞或成熟的巨噬细胞。 </li><li>转移培养上清中含有非贴壁祖到50毫升锥形猎鹰管和离心5分钟在1200转。 </li><li>重悬细胞沉淀IMDM培养5毫升，10％FCS和核细胞数在20乙酸稀释细胞悬液1。此过程中溶解的所有细胞，包括红血细胞和余下的核可以在血球计数。 </li><li>在浓度为0.5×10 6细胞/毫升完全培养基重悬细胞; IMDM培养，10％胎牛血清，150微米monothioglycerol胺酶（MTG），10毫微克/毫升要么MCSF，GM-CSF或IL-3。以MCSF衍生的巨噬细胞是经典而GM-CSF或IL-3-衍生的巨噬细胞激活或者激活的巨噬细胞。 </li><li>在4天更换培养基，纺非贴壁细胞，并把他们送回烧瓶，并在第7天，丢弃非贴壁细胞。 </li><li>此外，γ干扰素（10毫微克/毫升）或IL-4（10毫微克/毫升），可以添加到烧瓶包含歪斜巨噬细胞在第7天的派生MCSF-细胞经典激活或交替激活型，分别为。孵育3天的细胞。 </li><li>在10天，除去媒体和解除细胞组织培养瓶使用细胞分离液（GIBCO-BRL）。放置5毫升的缓冲5分钟，在室温（18℃）对细胞，然后与你的手掌根部牢牢几次一鼓作气瓶侧面。确保已解除，细胞通过检查下瓶瓶显微镜。到15毫升锥形猎鹰管吸取悬浮细胞，用IMDM培养，10％FBS的一个额外的10毫升的烧瓶。游泳池和降速在5分钟的300×G细胞。 </li><li>可行的使用浓度在10 7细胞/ ml无菌PBS PH7.4 1血细胞计数和重悬细胞计数。巨噬细胞注射到小鼠体内的准备。 </li><li>储备1.0×10 6巨噬细胞巨噬细胞表型的评估。经典激活巨噬细胞脂多糖分泌高浓度的一氧化氮和IL-10 IL-12p70和低水平的刺激相比，或者激活巨噬细胞。另外激活的巨噬细胞组成性表达YM1和精氨酸我（ArgI）的，可以由西方免疫检测。 </li></ol> 3。 Tranferring到小鼠的巨噬细胞<ol><li>在无菌PBS重悬巨噬细胞在10 7细胞/毫升的浓度。这允许在travenous注射巨噬细胞中的总体积100μL，这是一个可以安全，舒适的尾静脉注入小鼠的体积10 6。 </li><li>使用加热垫或热圈的温暖小鼠尾静脉扩张为5-10分钟。监视鼠标热疗的迹象倍。 </li><li>鼠标转让限制设备允许访问的尾静脉。 </li><li>尾部旋转90°，使静脉朝上。脉下来的尾巴两侧横向运行，并可以用来要么静脉。 </li><li>用酒精棉签清洁注射部位，并慢慢注入100μL，巨噬细胞（10 6），使用了26或28号针头的1 ml注射器。插入锥朝上，并在一个很小的角度，几乎平行的静脉针。 </li><li>如果正确插入针应感到无阻力和静脉注射后应成为明确。如果针是不是在静脉，尾巴会凸出。如果发生这种情况，取出针，再试最后一次尝试，或在其他静脉近端。每次注射必须使用一种新的针尾是艰难的，针尾静脉注射过程中迅速变得索然无味。 </li><li>注射后，取出针，适用于温和的压力，直到出血停止注射部位，并监视鼠标额外的5分钟，以确保出血已停止。 </li><li>每4天重复巨噬细胞注射到确保偏光巨噬细胞在实验过程中的连续输送。 </li><li>在实验结束后，随着实验分析，从网站的利益组织，并修复与多聚甲醛或甲醛确认极化的巨噬细胞的免疫组织化学染色的有效交付。 </li></ol> 4。代表结果居民macrop的数量和表型在任何特定的组织hages取决于被检查的组织，将改变在感染或炎症。同样，含有氯膦酸二钠脂质体的能力，从组织的巨噬细胞消耗，将取决于管理和有效的传递到靶组织路线。腹腔注射含有氯膦酸二钠脂质体在DSS诱导的结肠炎明显的巨噬细胞的标记，F4/80的（ 图1A）免疫组化染色过程中的小鼠结肠巨噬细胞在F4/80的减少+结果。也可以被用来组织化学染色，以确定在组织切片中的巨噬细胞数量和表型的数量差异。 图1B显示，没有发现在小鼠结肠枯竭相比，平均55％的巨噬细胞含有氯膦酸二钠脂质体耗尽时，老鼠被注射PBS仅作为喷射控制。每个值由计数F4/80的和ArgI染色编辑序列组织切片的阳性细胞从6小鼠在3分，3节每鼠标，计数被两个人失明的实验条件下进行。这些结果表明，腹腔注射氯膦酸二钠脂质体消耗在小鼠结肠巨噬细胞。 巨噬细胞从骨髓中存在不同的生长因子的产量是经典激活（MCSF-派生或，MCSF衍生与γ干扰素治疗），或者激活巨噬细胞（GM-CSF或IL-3-派生或MCSF派生和推导与IL-4处理）。法拉盛2从8周龄小鼠股骨和2胫骨骨髓产生鼠标6×10 7个有核细胞和产量8×10 6 MCSF源性巨噬细胞，10×10 6 GM-CSF源性巨噬细胞，或12× 10 6，IL-3-源性巨噬细胞。图2A显示IL-12p70/IL-10比在巨噬细胞和亚硝酸盐支持治疗与ernatants 图2B显示了一个典型的免疫印迹分析0.5×10 6的 MCSF巨噬细胞的+ / - 24小时内毒素，IL-4衍生条件与抗体或者激活的巨噬细胞标记，ArgI，YM1探测，或作为一个GAPDH的装载控制。这些数据表明，骨髓来源的巨噬细胞可以扭曲一个推导过程中的极化型。 体外的极化巨噬细胞的有效转移到组织是依赖于管理和组织路线。在DSS诱导的结肠炎，巨噬细胞注入静脉前往炎症部位 ​​和另外激活的巨噬细胞减少疾病的严重程度。 图3A显示巨噬细胞总数和组织切片计数小鼠ArgI +（或者激活）的巨噬细胞的数量与极化注入骨髓衍生的巨噬细胞。极化的巨噬细胞的影响转移到了在DSS诱导的结肠炎结肠疾病活动指数，体重减轻，直肠出血，减少粪便的一致性（ 图3B）根据添加剂得分。这些结果表明， 离体派生，继转移的巨噬细胞可以交通结肠和影响引起的炎症。不影响货币供应量M1巨噬细胞炎症，而M2巨噬细胞显着减少疾病活动指数。 <img src="/files/ftp_upload/4105/4105fig1.jpg" alt="图1"/> 图1。氯磷酸二钠，含有脂质体有效地消耗F4/80的+和F480的+ ArgI +细胞在小鼠体内冒号。给出一个混合的C57BL / 6×129Sv背景的野生型小鼠F2代7天，腹腔注射5％DSS的PBS（管制）或氯膦酸二钠含脂质体（氯磷酸二钠）4天前DSS处理0天，2，4和6 DSS处理过程中。冒号（一）被拆除，固定，切片，免疫组化染色为成熟的巨噬细胞（F4/80的）和M2巨噬细胞标记，ArgI。 （二）定量F4/80 + ArgI +巨噬细胞的。阳性染色细胞计数由蒙蔽倍率在40×6 /鼠标3个部分，从6小鼠/组领域的实验条件下的两个人。 * P &lt;0.01; <img src="/files/ftp_upload/4105/4105fig2.jpg" alt="图2"/> 图2。以 MCSF衍生的巨噬细胞表达经典激活型和IL-4治疗MCSF源性巨噬细胞表达或者激活的巨噬细胞表型。 （一）与经典激活的表型一致，MCSF源性巨噬细胞产生脂多糖，一氧化氮水平较高，并可以测量的的格里斯检测，检测其下游代谢产物，亚硝酸盐。他们ALSO产生更高的比例IL-12p70/IL-10，可以用ELISA法测定。 * N = 3，P &lt;0.01。 （二）或者激活的表型一致，MCSF-源性巨噬细胞，IL-4组成明示YM1和ArgI治疗，可以由西方免疫印迹检测。数据代表3独立的实验结果类似。 <img src="/files/ftp_upload/4105/4105fig3.jpg" alt="图3"/> 图3。静脉注射， 体外派生或者激活巨噬细胞结肠交通，减少肠道炎症。混合C57BL / 6×129Sv背景的野生型小鼠F2代六天分别给予5％的饮用水决策支持系统。 M1和M2巨噬细胞静脉注射DSS处理0和4天期间。冒号（一）被拆除，固定的，成熟的巨噬细胞（F4/80的）和M2巨噬细胞标记（ArgI）免疫组化染色切片。定量控制PBS注射小鼠（三）巨噬细胞，与货币供应量M1巨噬细胞（M1）余额（M2），M2巨噬细胞注入小鼠注射的小鼠进行计数阳性染色细胞在6个领域40×放大倍率从3节/鼠标从6只/组。计数是由两个人失明的实验条件。 （二）疾病活动的监测和治疗期间每天打进体重减轻，直肠出血，大便一致性下降，是一种添加剂得分。 *，P &lt;0.05为n = 6 2个独立的实验小鼠。

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没有利益冲突的声明。

巨噬细胞的吞噬细胞在免疫系统中发挥重要作用。他们是负责发起的先天免疫反应和指挥后天免疫反应。通常情况下，经典激活巨噬细胞被激活IFNγ的或LPS，并有责任为消除病原体和安装的炎症反应1。相反，IL-4和IL-13激活或者激活巨噬细胞和炎症的决议过程中发挥在碎片中的作用，清除和组织重塑。专门的巨噬细胞在肠道保留其杀菌活性，但不装入一个亲炎症反应2。这种有益菌?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>氯膦酸二钠，含脂质体</td><td> <a href="http://Clodronateliposomes.org" target="_blank">Clodronateliposomes.org</a> </td><td></td></tr><tr><td> PBS含脂质体</td><td> <a href="http://Clodronateliposomes.org" target="_blank">Clodronateliposomes.org</a> </td><td></td></tr><tr><td>无菌PBS pH7.4的</td><td> Invitrogen公司</td><td> 14190 </td></tr><tr><td> IMDM培养</td><td> Invitrogen公司</td><td> 12440 </td></tr><tr><td>胎牛血清（FCS）的</td><td> Invitrogen公司</td><td> 12483 </td></tr><tr><td>醋酸</td><td> BDH问题Aristar </td><td> BDH3092-500MLP </td></tr><tr><td> monothioglycerol胺酶（MTG） </td><td>西格玛</td><td> 96-275 </td></tr><tr><td> RECOMBinant小鼠MCSF </td><td>干细胞技术</td><td> 02951 </td></tr><tr><td>重组鼠GM-CSF的</td><td>干细胞技术</td><td> 02935 </td></tr><tr><td>重组小鼠IL-3 </td><td>干细胞技术</td><td> 02903 </td></tr><tr><td>重组小鼠γ干扰素</td><td>干细胞技术</td><td> 02746 </td></tr><tr><td>重组小鼠IL-4 </td><td>干细胞技术</td><td> 02714 </td></tr><tr><td>细胞分离缓冲器</td><td> Invitrogen公司</td><td> 13150-016 </td></tr><tr><td>大鼠anti-F4/80抗体</td><td> ABD Serotec公司</td><td> MCA497GA </td></tr><tr><td>鼠抗ArgI抗体</td><td> BD转导实验室</td><td> 610708 </td></tr></tbody></table> 表1。在本协议中使用的特定试剂。

depletion and reconstitution of macrophages in mice

巨噬细胞是重要的球员，在传染病的挑战或损伤的先天免疫反应，发起的先天免疫反应和指挥后天免疫反应。巨噬细胞功能障碍可能导致无法安装适当的免疫反应，因此，已在许多疾病的进程，包括炎症性肠病牵连。巨噬细胞显示极化表型大致分为两类。经典活化的巨噬细胞，与干扰素γ或LPS刺激激活，发挥了至关重要的作用，在细菌的挑战，而另外激活的巨噬细胞，IL-4和IL-13激活，参与清除杂物和组织重塑和已在该决议牵连阶段的炎症。在体内的炎症反应，巨噬细胞的免疫细胞浸润的复杂混合物中发现，并可能加剧或resolvin参加Ğ炎症。巨噬细胞的作用，在整个动物模型的定义在原地 ，这是必要的检查消耗从复杂的环境中的巨噬细胞的影响。极化的巨噬细胞表型定义问原位巨噬细胞表型的作用的问题，可以导出体外 ，从骨髓穿刺，并加回或没有事先枯竭的巨噬细胞，小鼠。在这里提出的协议中含有氯膦酸二钠，脂质体，与PBS注射控件，用来消耗结肠巨噬细胞在葡聚糖硫​​酸钠（DSS）诱导小鼠结肠炎。此外，巨噬细胞的极化，推导出体外，并转移到小鼠静脉注射。这种方法的一个需要注意的是，氯膦酸二钠含脂质体耗尽所有专业的吞噬细胞，包括树突状细胞和巨噬细胞，所以，以确保枯竭所观察到的效果是巨噬细胞特异性，重组ØF的巨噬细胞过继转移表型是必要的。全身巨噬细胞耗竭小鼠也可以通过回交小鼠实现到细胞CD11b-DTR背景，这是一个很好的补充办法。含有氯膦酸二钠脂质体介导的枯竭的优势是，它不参与回交小鼠需要的时间和费用，它可以在小鼠小鼠（C57BL / 6小鼠，BALB / c小鼠，或混合背景的背景下，无论使用）。

Watch this Scientific Journal Video about Depletion and Reconstitution of Macrophages in Mice at JoVE.com

Depletion and Reconstitution of Macrophages in Mice

巨噬细胞发挥核心作用的动态平衡，并在许多组织的病理。这里介绍的协议描述消耗巨噬细胞的方法<em&gt;在体内</em&gt;，来自偏光巨噬细胞，骨髓穿刺，继转移到小鼠巨噬细胞。这些技术允许极化的巨噬细胞在健康和疾病中发挥的作用的决心。

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

depletion-and-reconstitution-of-macrophages-in-mice

Research

JoVE Journal

Immunology and Infection

39.4K 次观看.  University of British Columbia. 巨噬细胞发挥核心作用的动态平衡，并在许多组织的病理。这里介绍的协议描述消耗巨噬细胞的方法在体内，来自偏光巨噬细胞，骨髓穿刺，继转移到小鼠巨噬细胞。这些技术允许极化的巨噬细胞在健康和疾病中发挥的作用的决心。

视频: Depletion and Reconstitution of Macrophages in Mice

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

调整降解以实现特定和高效的蛋白质消耗

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...