资助这项研究是由加拿大血液服务（CBS）和加拿大研究所（CIHR）健康科学的研究合作基金。作者感谢的LMB高分子枢纽的UBC血液研究中心的研究设施的使用。基础设施的支持，加拿大创新基金会（CFI）和迈克尔史密斯健康研究基金会（MSFHR）。河Chapanian是一个收件人（CIHR / CBS）在输血科学博士后奖学金和收件人的MSFHR研究实习生博士后奖学金。 JN Kizhakkedathu是一个收件人的MSFHR生涯研究学者奖。

A.超支化的聚甘油改造（SS-HPG） <ol><li>地点冻干HPG 60 kDa的（0.5克，0.0083毫摩尔）在一个圆底烧瓶中，并真空下干燥过夜，在90℃下</li><li>冷藏烧瓶至室温，并溶解在无水吡啶（3ml）中的干燥的HPG。 </li><li>若要官能约8个羟基基团与羧基基团上HPG，添加催化量的二甲基氨基吡啶（5毫克/毫升的吡啶溶液一滴）的HPG溶液。到该混合物中，加入琥珀酸酐，（0.0067克，0.0664毫摩尔）溶解在0.5毫升吡啶在10分钟内逐滴。在氩气下在室温下搅拌该混合物过夜。 </li><li>沉淀的混合物于40毫升冷的丙酮（4℃），在50毫升的离心管中，离心使用Beckman J2-MC离心机以27,000 xg离心15分钟。滗析出上清液，并除去​​残留的丙酮中，在室温下用氩气冲洗。 </li><li>要激活的羧基升用琥珀酰亚胺琥珀酸酯（SS）的基团的基团，溶解在3ml无水DMF中的羧基官能化的HPG。加入N-羟基琥珀酰亚胺（0.0077克，0.0664毫摩尔）和N，N&#39;-二异丙基碳二亚胺（0.0084克，0.0664毫摩尔）的HPG溶液，并在氩气下在室温下搅拌该混合物过夜。 </li><li>冷丙酮中沉淀，净化SS-HPG。 </li><li>除去残留的丙酮中，通过用氩气冲洗。 </li><li>改性的HPG和羧基和SS的官能化程度的质子（1 H） - NMR分析确定的纯度。 </li></ol> B.全血的收集和分离的红血细胞（红细胞） <ol><li>收集全血（40％的血细胞比容，3毫升），从同意健康的人供体的成柠檬酸vacutainer管的。 </li><li>离心柠檬酸管在1,000 xg离心4分钟。 </li><li>除去上清液，含有血浆和血小板，和中间的血沉棕黄层，包含白血细胞和platele日ts使用巴斯德吸管。 </li><li>转移到一个12毫升的猎鹰离心管中的填充的红细胞（血细胞比容80％，1.2毫升），并添加PBS缓冲液（8毫升，pH值= 8.0）洗红细胞。 </li><li>通过反演获得细胞分布均匀混合的红细胞。 </li><li> Falcon管离心在1000 XG 4分钟，去除上清。 </li><li>洗净红细胞用PBS两次通过重复步骤5和6。 </li><li>地点包装-红细胞（100微升）在1.6 ml Eppendorf管中，添加PBS缓冲液（300微升，pH值= 8.0），得到20％的血细胞比容的红细胞。 </li></ol> C.红细胞HPG嫁接<ol><li>后立即沉淀变形的HPG在丙酮中，在步骤A6中，地点SS-HPG（150毫克），在玻璃小瓶（1打兰），和溶解在PBS缓冲液（300微升，pH值= 8.0）。 </li><li> SS-HPG的聚合物溶液添加到20％的血细胞比容洗涤红细胞，得到最终体积为400μl和SS-HPG浓度，取值范围从0.5毫米到3毫米。例如，准备用0.5毫米的红细胞SS-HPG，取出36微升PBS洗涤后的红细胞和SS-HPG的聚合物溶液放置36微升。 </li><li>涡的悬浮液轻轻Eppendorf管中放置1小时，在室温下轨道摇床。 </li><li>离心的Eppendorf管中，以1,000×g离心4分钟，并移液上清液。 </li><li>加入1ml的PBS洗红细胞，离心并除去上清液。 </li><li>加入1 ml生理盐水和清洗红细胞的两倍，在步骤5中。 </li><li>加入300μl的生理盐水100μl的包装红细胞（血细胞比容80％）至终浓度为20％的血细胞比容。 </li></ol> D.表征丘脑 - 垂体 - 性腺修改红细胞一，补体介导的裂解<ol><li>收藏报错全血的8毫升左右从健康的人供体到BD真空容器玻璃血清管，并在室温下搅拌30分钟，使血液凝块。 </li><li>离心管在2,000 xg离心10分钟，并收集3毫升血清左右。 </li><li> 1毫升的SERU米，在60℃下在水浴中放置30分钟以制备热灭活血清。 </li><li>新鲜血清或热灭活的血清中加入60μl到Eppendorf管中，包含60微升20％的血细胞比容的HPG改性红细胞或未修改的红细胞。 </li><li>孵育红细胞1小时在37℃下</li><li>要量化的细胞悬浮液中的血红蛋白量，5.9微升20％的血细胞比容的HPG改性红细胞或未修饰的细胞在96孔板的一式三份。添加294微升德拉布坎试剂（试剂，用于量化的血红蛋白量spectrophotomerically）。德拉布坎的试剂混合细胞通过移液中出。测量使用SPECTRA MAX 190板读数器在540 nm处的吸光度的血红蛋白cyanoderivati​​ve的。 </li><li>为了定量上清液中的血红蛋白量，离心细胞以13,000 xg离心1分钟。将50μl的上清液在96孔板的一式三份。加入250μl德拉布坎的试剂。混合细胞与德拉布坎的试剂通过移液中和了。测量使用SPECTRA MAX 190板读数器在540 nm处的吸光度的血红蛋白cyanoderivati​​ve的。 </li><li>量化裂解细胞的量，从上清液中血红蛋白的比率（步骤7）和样品中的总的血红蛋白（步骤6），8，11，12。 </li></ol> II。主要和次要抗原使用MTS牌的伪装<ol><li>将50微升的HPG改性的RBC（血细胞比容10％）在Eppendorf管中，离心机在1000×g离心1分钟，并除去上清液。 </li><li>加入110微升的MTS稀释剂的细胞沉淀，并通过移液和细胞悬浮液均匀。 </li><li>将11微升的细胞悬浮液添加到每个微型硅胶柱。避免接触凝胶在另外。 </li><li>找到MTS卡，成离心机卡持有人，并在156 XG离心6分钟。 </li><li>保护表面抗原从RBC在迷你硅胶柱的位置，测定基础上制造R描述。 </li></ol> III。双水相分割的测量<ol><li>准备葡聚糖500 kDa的/ PEG 8 kDa的两相分离系统（5％葡聚糖500k的，4％PEG 8K，150mM的NaCl，10mM磷酸盐）根据13组成。 </li><li>在433×g离心10分钟，在室温下离心，得到的两相体系（葡聚糖在底部）。 </li><li>仔细分开的两个阶段。 </li><li>上PEG阶段，进入一个的标记管（凹底）分装1.0毫升，加入20μl的20％HPG修饰RBC红细胞压积和混合细胞轻轻弹。 </li><li>加入0.5毫升的上层相，包含细胞低级相0.5毫升，并轻轻地混合系统由轻拂。 </li><li> 〜2分钟的系统分离，并确定细胞的位置，在系统中的管放置在板凳上。本地化的红细胞（PEG相的上部，在下部的葡聚糖相或两个）是函数的程度的修饰和分子重量HPG。 </li></ol> IV。渗透的脆弱测量的<ol><li>准备NaCl溶液的浓度范围从0到0.9（重量/体积）％。 </li><li>地点NaCl溶液0.4毫升1.5毫升的Eppendorf管。 </li><li>加入20μl的红细胞（血细胞比容20％）的NaCl溶液中。 </li><li>混合细胞仔细的反转，并将其放置在37℃的水浴中30分钟。 </li><li>仔细悬浮细胞，通过倒置。取50微升RBC悬浮液，并把它添加到1毫升德拉布坎的试剂，放置在一个反应​​杯中。德拉布坎的试剂混合细胞通过移液中出。测量的吸光度的血红蛋白cyanoderivati​​ve的，在540nm处，用BECKMAN COULTER DU 730 UV / VIS读者。 </li><li>为了定量上清液中的血红蛋白量，离心细胞在1,000×g离心1分钟。加入200μl的上清液到1毫升德拉布坎的试剂中的比色皿。德拉布坎的试剂混合细胞通过移液中出。测量吸光度的hemogl的在540 nm处，用BECKMAN COULTER DU 730 UV / VIS读者的obin cyanoderivati​​ve。 </li><li>计算从上清液中的量血红蛋白的比率（步骤6）中的细胞悬浮液（步骤5）的总的血红蛋白量不同的NaCl溶液中溶解的量的细胞。 </li></ol>五，流式细胞仪测量 - 保护恒河猴-D抗原（RHD） <ol><li>添加5μl的控制或的HPG改性红细胞（20％血细胞比容）补充到总体积为25μl的0.5％BSA的PBS缓冲液，一式三份。 </li><li>加入25μLFITC单克隆抗恒河猴D（RHD）购买商Biodiagnostics（PA，USA）。 </li><li>在水浴中，在37℃下孵育30分钟的混合物。 </li><li>洗净的混合物用1.5ml PBS缓冲液两次。离心1,000 xg离心1分钟，以除去上清液。 </li><li>暂停红细胞1 ml生理盐水，暂停并转移到4毫升流BD流式细胞仪检测管。 </li><li>分析使用FACSCanto II FLO瓦特流式细胞仪，获得5000 RBC控制栅极上的事件，并使用整个的事件进行分析。 </li></ol> VI。流式细胞仪测量 - CD47的表达<ol><li>添加1.54微升的控制或的HPG改性红细胞一式三份补充到总体积为44微升的0.5％BSA的PBS缓冲液（20％血细胞比容）。 </li><li>加入6微升藻红蛋白（PE）标记的小鼠抗人CD47购自BD Biosciences公司（新泽西州，美国）。 </li><li>在水浴中，在37℃下孵育30分钟的混合物。 </li><li>洗净两次用1.5ml PBS缓冲液的混合物。离心1,000 xg离心1分钟，以除去上清液。 </li><li>暂停红细胞1 ml生理盐水，通过26 G 5/8针，以减少细胞结块。 </li><li>暂停转移到4毫升流BD流式细胞仪检测管。 </li><li> FACSCanto II流式细胞仪分析使用，获得10000个事件的RBC控制门。 </li></ol>

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万能供血者红细胞有很大的潜力，在提高血液的输血治疗的有效性和安全性。红细胞也被认为是有前途的药物运输车辆，由于其长循环和固有的生物相容性，14日，15。本文提出的实验评估丘脑-垂体-性腺修改红细胞在体外的特点。 体外性能和丘脑-垂体-性腺修改红细胞在体内循环，本集团已在最近8，11。 Dextran500K/PEG8K双水相体系中，补充盐和钠磷酸盐，红细胞...

<table border="1"><tbody><tr><td>环氧丙醇</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td>三羟甲基丙烷</td><td> Fluka公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td>甲醇钾</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td>无水吡啶</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td> 4 - 二甲氨基吡啶</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td>丁二酸酐</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td>丙酮</td><td> Fisher Scientific则</td><td></td><td> （ON，加拿大） </td></tr><tr><td>无水二甲基甲酰胺</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td> N-羟基琥珀酰亚胺</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td> N，N&#39;-二异丙基碳二亚胺</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td> MTS卡</td><td></td><td></td><td>高输入系统（MTS）卡（FL，USA） </td></tr><tr><td>右旋糖酐500 kDa的</td><td> Pharmacia精细化学公司</td><td></td><td> （瑞典） </td></tr><tr><td> PEG 8 kDa的</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td> FITC单克隆抗恒河猴D（RhD血型） </td><td>商Biodiagnostics </td><td></td><td> （宾夕法尼亚州，美国） </td></tr><tr><td> PE单克隆抗-CD47 </td><td> BD Biosciences公司</td><td></td><td> （新泽西州，美国） </td></tr><tr><td>德拉布坎试剂</td><td> Sigma Aldrich公司</td><td></td><td> （ON，加拿大） </td></tr><tr><td></td><td></td><td></td><td> 表化学品和试剂，用于嫁接丘脑-垂体-性腺聚合物RBC膜并对其进行分析。 </td></tr></tbody></table>

antigens protected functional red blood cells by the membrane grafting of compact hyperbranched polyglycerols

红细胞（RBC）输血是用于治疗急性和慢性的医疗问题，如重型地中海贫血和镰状细胞贫血1-3一些至关重要的。由于众多的RBC表面上的（〜308已知抗原4），在慢性输血治疗的患者发展由于丢失如同输血红细胞4，5的轻微上的抗原的同种抗体的抗原的存在下。接枝亲水性聚合物，如聚乙二醇（PEG）和超支化聚甘油（HPG）的形成，RBC膜排除层上防止表面抗原的抗体的相互作用，而不会影响通过小分子如氧，葡萄糖，和离子3。目前，没有一种方法是可用于普遍的红血供体细胞的产生部分是因为大量抗原（基于蛋白质和碳水化合物）的存在下对RBC表面和d提出严峻的挑战才有发展这种方法将大大提高输血的安全性，并显着提高红细胞的可用性和使用。在这份报告中，提出的实验，是用来开发抗原保护功能红细胞由HPG及其表征膜接枝。 HPGs高度生物相容性紧凑的聚合物6，7，并预期位于小区内的的糖萼围绕的脂质膜8，9和掩模RBC表面抗原10，11。

迷彩恒河猴D抗原和CD47 RBC表面蛋白的流式细胞仪定量，使用荧光标记的单克隆抗体，和具有代表性的结果在图1中给出。在HPG-接枝红细胞的情况下（灰色），该信号的强度降低（峰转移至左）相比，控制红细胞（红色和绿色），表示表示掩蔽的表面蛋白的细胞表面结合的抗体减少。 <img src="/files/ftp_upload/50075/50075fig1.jpg" alt="图1" fo:content-...

Watch this Scientific Journal Video about Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols at JoVE.com

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

细胞膜的变形例的红血细胞（红细胞），超支化聚甘油（HPG）。修改红细胞进行了表征双水相分割，渗透脆性和补体介导的溶解。使用流式细胞仪和微的打字系统（MTS）的血液分型卡的表面蛋白抗原的伪装进行了评估。

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

antigens-protected-functional-red-blood-cells-membrane-grafting

Research

JoVE Journal

Immunology and Infection

18.0K 次观看.  University of British Columbia. 细胞膜的变形例的红血细胞（红细胞），超支化聚甘油（HPG）。修改红细胞进行了表征双水相分割，渗透脆性和补体介导的溶解。使用流式细胞仪和微的打字系统（MTS）的血液分型卡的表面蛋白抗原的伪装进行了评估。

视频: Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

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

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

定量检测抗体诱导补体激活的方法对红血细胞

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...