这项工作是支持该司院内研究的美国国家过敏和传染病研究所（NIAID），国立卫生研究院（NIH），美国。 所有小鼠均保持在美国协会认可的实验室动物护理动物设施在国家过敏和传染病研究所（NIAID）的评审，并根据实验室动物护理和使用指南中列出的程序安置在的主持下，由动物护理和使用委员会NIAID批准的协议。

1。小鼠骨髓细胞的分离<ol><li>安乐死小鼠使用该机构的动物保健委员会批准的协议，和动物表面喷用70％的乙醇。 </li><li>在中腹部的皮肤做一个切口，去除皮肤的鼠标从远端部分包括下肢皮肤覆盖。 </li><li>由下肢用剪刀切断肌肉和小心，同时避免破坏股骨头从髋关节脱位的髋臼。 </li><li>取出剩余使用手术刀和剪刀从股骨和胫骨的肌肉和上面的膝关节多加小心，不破骨端部从胫骨股骨分离。放置在培养皿中含有冰冷的RPMI 1640 1X补充有10％FBS和1％青霉素/链霉素的骨头。 </li><li>组织培养罩下，继续进行下面的步骤。采取额外的预防措施，以保持严格的无菌技术，以避免中性粒升激活。 </li><li>用70％乙醇（陪替氏培养皿内），然后由三个随后，在冰冷的无菌PBS洗涤（陪替氏培养皿内）从骨骼的表面冲洗掉乙醇冲洗每个骨骼。 </li><li>里面干净的无菌培养皿中，切断的骨骺的骨头，让他们一边。 </li><li>使用25号针头和一个12毫升注射器填充用的RPMI补充有10％FBS和2mM EDTA，并冲洗到在50ml的螺旋盖的Falcon管中装配有100μm的骨轴两端的骨髓细胞过滤。为了有效地去除所有细胞，刮去的内表面上使用25号针头的骨头。 </li></ol>注:骨骼热烫表明，细胞已被充分地刮掉。 注:使用冲洗股骨/胫骨对约10毫升的媒体。加入EDTA介质是必要的，以防止细胞聚集。 <ol start="9"><li>切骨骨骺的小0.5-1毫米手术刀和粉碎他们通过100微米的过滤器使用了2.5 ml的Eppendorf Combitip中加BIOPUR的枪头，后端3件。 </li><li>在4℃下离心7分钟1400转</li><li>裂解红血细胞重新悬浮细胞沉淀在20毫升0.2％NaCl溶液在约20秒，然后加入20毫升1.6％NaCl溶液。 （关键:不要超过20-30秒低渗裂解避免骨髓细胞死亡。低渗氯化钠溶解，建议在使用ACK裂解缓冲液，因为后者有可能激活中性粒细胞）。 </li><li>在4℃下以1400 rpm离心7分钟，以收集细胞。 </li><li>补充有10％FBS和2mM EDTA的RPMI 1640 1X洗涤细胞，并在步骤1.12中，离心。 </li><li>骨髓细胞，采用这种方法的收益率是未感染8-12周龄C57BL / 6小鼠约60-80亿美元。 </li></ol> 2。中性粒细胞的分离通过密度梯度离心<ol><li>计数骨髓细胞，并重新悬浮于1毫升冰冷的无菌PBS中。 </li><li>加入3毫升HISTOPAQUE 1119（密度，1.119克/毫升）在15毫升锥形管。 </li><li>覆盖层3 HISTOPAQUE 1077毫升（密度，1.077克/毫升）上的3毫升HISTOPAQUE 1119。 </li></ol>注:HISTOPAQUE 1119和1077 HISTOPAQUE的，应在使用前加热至18-26°C。 关键的步骤:准备即将使用前预先制备的梯度将导致在两个层之间的扩散和次优的嗜中性粒细胞的纯度和回收率的梯度。 关键的一步:重叠HISTOPAQUE的1077超过1119需要缓慢地进行，以避免混合两种密度，这将阻止细胞在离心分离。 <ol start="4"><li>叠加的骨髓细胞悬浮液的顶部HISTOPAQUE 1077。 </li></ol>关键的一步:承的过载阿英骨髓细胞悬液缓慢地进行，以避免干扰细胞之间的接口和HISTOPAQUE 1077在1077 HISTOPAQUE需求。 注意:从一个未感染的小鼠的骨髓细胞重新悬浮在1ml的PBS中产生中性粒细胞的纯度&gt; 90％。然而，汇集许多骨髓样本损害中性粒细胞的纯度，例如，300×10 6个细胞重新悬浮在3毫升PBS降低嗜中性粒细胞的纯度&gt; 90％〜80％。因此，调查人员应进行试点实验，以确定理想的细胞计数/体积为他们的具体的实验条件<ol start="5"><li> 30分钟2,000转25°C时不带制动离心机。 </li></ol>注:有效地分离的嗜中性粒细胞在室温下梯度离心是至关重要的，必需的。 <ol start="6"><li>收藏HISTOPAQUE 1119的界面处的嗜中性粒细胞HISTOPAQUE 1077层。 </LI&gt; <li>洗两次收集到的嗜中性粒细胞用RPMI 1640 1X补充有10％FBS和1％青霉素/链霉素和离心7分钟，在1400 rpm的转速下，在4℃下</li><li>嗜中性粒细胞计数和确定其可行性。 </li></ol>注:中性粒细胞通常&gt; 95％是可行的，通过流式细胞仪分析纯度&gt; 90％确定。典型的产量与未受感染8-12周龄C57BL / 6小鼠中性粒细胞从骨髓（ 即 2股骨和胫骨骨头）约6-12亿个细胞。这个数字是大大提高中性粒细胞收获时从受感染的动物的骨髓。因此，〜30-40万中性粒细胞/鼠标念珠菌感染小鼠用于细胞收集6时恢复。 3。中性粒细胞使用CellTracker染料标记<ol><li>重悬嗜中性粒细胞以5×10 6细胞/ ml的PBS中在37℃下预热</li><li>添加一个CellTracker染料最终concent5微米的口粮。 </li></ol>注:CellTracker绿色（CMFDA（5 - 氯甲基二乙酸酯）和（CMTMR CellTracker橙（5 - （6）4 - 氯苯甲酰氨基TETRAMETHYLRHODAMINE的）使用在这个协议中差异标记的嗜中性粒细胞从野生型和基因缺失型小鼠。 注:准备原液10毫米CellTracker绿色和橙色CellTracker，分装和储存在-80°C的实验，直到有一天。 <ol start="3"><li>孵育5μM的的相应CellTracker染料在黑暗中振荡水浴中10分钟，在37℃的中性粒细胞。 </li><li>洗涤细胞两次，用冰冷的RPMI 1640 1X补充有10％FBS和1％青霉素/链霉素。 </li></ol>注:高效的洗涤后的细胞的标记步骤是必不可少的与CellTracker染料染料，以避免交叉污染混合之前的差异标记的嗜中性粒细胞种群起伏tream有竞争力的复育研究。 4。继转移小鼠中性粒细胞和转移中性粒细胞，使用流式细胞仪分析<ol><li>中性粒细胞重悬在冰冷的PBS中的浓度为25×10 6细胞/ ml，200μl的悬浮液注入到尾静脉中，让每只小鼠5×10 6个中性粒细胞转移。具有竞争力的再增殖嗜中性粒细胞的研究中，野生型基因缺失的嗜中性粒细胞，按1:1的比例混合，并注入5×10 6每只小鼠嗜中性粒细胞的共如上。 </li></ol>注:至少提高至10×10 6嗜中性粒细胞过继转移每只小鼠无明显立即对动物的毒性。 <ol start="2"><li>继转移（ 例如，1，2，3或4小时后转移）后，在不同的时期，安乐死小鼠收获的血液，和/或骨髓和/或其他靶器官（次）的兴趣。 </li><li>准备SIngle细胞悬液继转移使用协议公布6,15标记的嗜中性粒细胞的定量和定性分析，从这些组织。 </li><li>死/活的可行性染色和FC封锁，标记细胞CD45（克隆30-F11），Ly6G（克隆1A8）和CD11b（M1/70克隆）和栅极活CD45 + Ly6G +细胞CD11b +中性粒细胞。在此栅极的嗜中性粒细胞包括原生嗜中性粒细胞的受体小鼠中，以及过继转移的标记的嗜中性粒细胞，FITC +（如标记与CellTracker格林）或PE（如标记的的细胞跟踪奥兰治。）。 </li></ol>注:中性粒细胞在血液，骨髓和肾念珠菌感染小鼠跟踪至少4小时后转移。 注:用2％多聚甲醛的PBS固定不产生不利影响的标记与CellTracker石墨的嗜中性粒细胞的平均荧光强度EEN或CellTracker的橙色至少48小时。

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作者宣称，他们有没有竞争的利益。

在这里，我们提出了一个可靠，简单，快速，经济的协议，用于隔离大量嗜中性粒细胞从骨髓的小鼠具有纯度高，使用密度梯度离心法的可行性。 〜6-12×10 6中性粒细胞正确执行此协议，可以从收回未受感染的小鼠，从小鼠在感染后6和多达〜30-40×10 6中性粒细胞可能被孤立。分离出的嗜中性粒细胞是80-95％的纯&gt; 95％是可行的。 骨髓是一种合适的水库...

嗜中性粒细胞是人类最丰富的白细胞。他们是主要的先天免疫系统和抵御入侵的微生物的第一线作为细胞成分。获得性中性粒细胞减少症和原发性免疫缺陷的患者，影响中性粒细胞数量和/或功能的发展危及生命的侵袭性细菌和真菌感染，这些细胞在宿主防御1突出的重要性。在一场精心策划的先天免疫反应，从而导致分泌趋化因子产生趋化梯度能够招募中性粒细胞从血液中进入发炎组织2诱导其同源的模式识别受体的结果入侵的病原体在感染部位的免疫识别。中性粒细胞进入感染部位后，他们成为激活，导致细胞因子和趋化因子的生产，病原体吸收，并通过氧化和非氧化我杀chanisms 3。除了 ​​大家公认的先天免疫的作用，中性粒细胞也被最近表现出作为有效的适应性免疫反应的发起人，扮演着重要的角色。另一方面，从它们的免疫保护作用外，中性粒细胞也可介导组织损伤和由于过度积累和/或激活在炎症部位 ​​的免疫病理学，如图中的各种感染和自身免疫性疾病5-7。 尽管中性粒细胞在安装有效的先天免疫反应和一些传染性疾病和炎症性疾病，技术上的困难，处理这些细胞，并缺乏可靠的实验协议的多效性效应功能不可或缺的作用受阻的研究，在过去的几十年里，中性粒细胞。因此，应进一步促进使用可重复的检测，隔离中性粒细胞中性粒细胞介导的​​immunolo研究gical功能体外和体内 。迄今为止，已经描述的几种方法已用于分离，如密度梯度离心的人血和小鼠血液或骨髓8,9，正极或负极的从小鼠血液或骨髓10,11中的嗜中性粒细胞的免疫磁性富集的嗜中性粒细胞，和收获巯基乙酸或其他抗炎剂12后，腹腔注射的小鼠的腹腔内中性粒细胞。虽然可以很容易地从人血中分离出大量中性粒细胞，这种方法是最理想的小鼠由于数量有限小鼠血液，排除隔离足够的中性粒细胞功能的研究或过继转移实验13。此外，虽然产量的巯基乙酸盐诱导的腹膜腔中的细胞相比，小鼠血嗜中性粒细胞在炎症腹腔灌洗，纯度之间变化60-90％，分离出的嗜中性粒细胞，表现出一个激活的表型。因此，收集的细胞用这种方法只能用于执行功能的激活的研究，但并不代表未受刺激的嗜中性粒细胞，小鼠腹膜腔有中性粒细胞数，在稳定状态12。相反，骨髓是一个方便的水库收获大量的任何未受刺激或激活的中性粒细胞11,14，它可以被用于下游功能性研究，如吞噬作用，杀害和脱颗粒，或过继转移到受体小鼠。 在这里，我们介绍一个简单而快速的协议（约2小时），它提供了一个高收益率（〜6-12×10 6中性粒细胞/未受感染的鼠标，或可达30-40×10 6中性粒细胞/感染小鼠）纯（80 -95％）嗜中性粒细胞&gt; 95％的存活率，从骨髓。此方法使用市售HISTOPAQUE，密度梯度细胞分离配给介质组成的聚蔗糖和泛影葡胺钠，分离从小鼠骨髓中的嗜中性粒细胞。此方法产生的显着更大的每只小鼠的嗜中性粒细胞的数目相比，血液或腹膜腔，它可以被用来收集在稳定状态或感染后的小鼠嗜中性粒细胞，它是比较容易的层相比，使用不连续的密度梯度离心法Percoll密度梯度组成的55％/ 65％/ 75％的Percoll 9在PBS中。此外，收集纯净的中性粒细胞所需的时间和资源相比显着下降，中性粒细胞隔离，利用荧光激活细胞分选。此外，因为这种方法不涉及免疫磁性富集步骤，更具成本效益的，它避免了细胞暴露磁性柱和抗体，从而减少中性粒细胞活化的可能性。 在除了执行功能隔离neutroph研究中ILS 体外和细胞过继转移到受体小鼠，该协议还描述了一种方法，用于标识分离的嗜中性粒细胞，使用不同的CellTracker染料。从不同遗传背景的小鼠中性粒细胞的鉴别标签跟踪转移的嗜中性粒细胞在受体小鼠的组织，采用流式细胞仪，它可以提供机械洞察力贩运中性粒细胞从血液中特定基因的直接作用，可适应在竞争激烈的复育研究进入靶器官发炎6。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>RPMI 1640 1X with L-glutamine and 25 mM HEPES</td>
 <td>Cellgro</td>
 <td>10-041-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (Heat inactivated)</td>
 <td>GemCell</td>
 <td>100500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)</td>
 <td>GIBCO</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M EDTA</td>
 <td>Quality Biological Inc</td>
 <td>351-027-101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.2% and 1.6% sodium chloride</td>
 <td>JT Baker</td>
 <td>3624</td>
 <td>Sodium chloride solutions prepared using distilled water and sterile filtered</td>
 </tr>
 <tr>
 <td>Sterile filtered Histopaque 1077</td>
 <td>Sigma</td>
 <td>10771</td>
 <td>Histopaque 1077 needs to be brought to 18-26 &deg;C before use</td>
 </tr>
 <tr>
 <td>Sterile filtered Histopaque 1119</td>
 <td>Sigma</td>
 <td>11191</td>
 <td>Histopaque 1119 needs to be brought to 18-26 &deg;C before use</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline (PBS) without Calcium and Magnesium</td>
 <td>Cellgro</td>
 <td>210-40-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethyl Alcohol (200 proof)</td>
 <td>The Warner Graham Company</td>
 <td>64-17-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CellTracker Green (CMFDA, 5-Chloromethylfluorescein Diacetate)</td>
 <td>Invitrogen</td>
 <td>C-7025</td>
 <td>Make stock solutions of 10 mM in DMSO, aliquot and store at -80 &deg;C</td>
 </tr>
 <tr>
 <td>CellTracker Orange (CMTMR, 5-(and 6)-4-Chloromethyl Benzoyl Amino Tetramethylrhodamine)</td>
 <td>Invitrogen</td>
 <td>C-2927</td>
 <td>Make stock solutions of 10 mM in DMSO, aliquot and store at -80 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-mouse CD45 (Clone 30-F11)</td>
 <td>eBioscience</td>
 <td>170451-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse Ly6G (Clone 1A8)</td>
 <td>BD Pharmingen</td>
 <td>551461</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse Ly6G (Clone 1A8)</td>
 <td>BD Pharmingen</td>
 <td>560599</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b (Clone M1/70)</td>
 <td>eBioscience</td>
 <td>47-0112-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse CD16/CD32 (Mouse BD Fc Block)</td>
 <td>BD Pharmingen</td>
 <td>553141</td>
 <td>Use at 1:100 dilution</td>
 </tr>
 <tr>
 <td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
 <td>Molecular Probes</td>
 <td>L-23105</td>
 <td>Use at 1:1000 dilution</td>
 </tr>
 <tr>
 <td>Dimethyl Sulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>67-68-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde Solution, 4% in PBS</td>
 <td>USB Corporation</td>
 <td>19943</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table 1. Reagents for isolation, labeling and tracking of adoptively transferred neutrophils.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>C57BL/6 mice</td>
 <td>Taconic</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes</td>
 <td>Corning</td>
 <td>430053</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml centrifuge tubes</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>25 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229225B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229210B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5 ml serological pipettes</td>
 <td>Celltreat</td>
 <td>229205B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pasteur pipettes (3 ml)</td>
 <td>BD Falcon</td>
 <td>357575</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>12 ml syringes</td>
 <td>Kendall monoject</td>
 <td>512878</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>25 G x 5/8 in. Needles (precision glide needles)</td>
 <td>BD</td>
 <td>305122</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 mm cell strainers</td>
 <td>BD Falcon</td>
 <td>352360</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bactericidal Petri dishes</td>
 <td>BD Falcon</td>
 <td>351029</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Combitips Plus Biopur</td>
 <td>Eppendorf</td>
 <td>2249608-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse dissecting instruments (Scissors, forceps, scalpel)</td>
 <td>Biomedical Research Instruments</td>
 <td>10-2300, 10-2165, 25-1200, 26-1000</td>
 <td>Instruments sterilized prior to use</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Tissue culture hood</td>
 <td>The Baker Company</td>
 <td>SG403</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Refrigerated centrifuge</td>
 <td>Thermo Fischer Scientific</td>
 <td>75004521</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>37 &deg;C shaking water bath</td>
 <td>Thermo Fischer Scientific</td>
 <td>3166721</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table 2. List of Materials and Equipment used in this protocol.</td>
 </tr>
 </tbody>
</table>

isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments

嗜中性粒细胞是关键的先天免疫系统的效应细胞。他们正在迅速招募急性炎症部位，发挥保护或致病作用取决于炎症环境。然而，尽管中性粒细胞免疫力，详细了解调解中性粒细胞的效应，在不同的传染性疾病和炎症性疾病的免疫病理作用的分子因素，不可或缺的作用仍然缺乏，部分原因是由于其半衰期短，处理这些困难细胞和缺乏可靠的实验协议，获得足够数量的下游功能研究和中性粒细胞过继转移实验。因此，操作简单，快速，经济，可靠的方法是非常可取的收获足够数量的鼠标中性粒细胞的吞噬功能，杀灭，细胞因子的产生，脱粒和贩运等功能评估。为此目的，我们提出了可再现的密度梯度离心法为基础的协议，它可以适应于任何的实验室分离大量的中性粒细胞从小鼠骨髓中的具有高纯度和活力。此外，我们提出了一个简单的协议，使用CellTracker染料标记隔离中性粒细胞，然后可以过继转移到受体小鼠在一些组织和跟踪至少4个小时的转移后，使用流式细胞仪。使用这种方法，可以成功地采用了差异化标记的嗜中性粒细胞从野生型和基因缺陷小鼠不同CellTracker染料，执行竞争力的评价贩卖中性粒细胞从血液中特定基因的直接作用到在体内靶组织的再增殖研究。

这个协议被优化用于从小鼠骨髓细胞的收获和随后的分离的嗜中性粒细胞通过密度梯度离心使用市售HISTOPAQUE细胞分离介质从这些细胞。嗜中性粒细胞分离，使用这种方法可用于多种下游功能性研究体外受体小鼠过继转移实验中。 未受感染8-12周龄C57BL / 6小鼠双侧股骨和胫骨的骨髓细胞，每收集典型的产量为60-80万〜〜94-98％具有可行性细胞。下行密度梯度离心这些细胞?...

Watch this Scientific Journal Video about Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments at JoVE.com

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

我们描述了一个协议，用于分离和纯化从小鼠骨髓中的嗜中性粒细胞通过密度梯度离心和嗜中性粒细胞标记使用CellTracker染料。这是一个简单，快速，重复性好，经济的方法获得大量的嗜中性粒细胞的下游功能研究或过继转移和跟踪实验。

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

isolation-purification-labeling-mouse-bone-marrow-neutrophils-for

Research

JoVE Journal

Immunology and Infection

61.5K 次观看.  National Institute of Allergy and Infectious Diseases, NIH. 我们描述了一个协议，用于分离和纯化从小鼠骨髓中的嗜中性粒细胞通过密度梯度离心和嗜中性粒细胞标记使用CellTracker染料。这是一个简单，快速，重复性好，经济的方法获得大量的嗜中性粒细胞的下游功能研究或过继转移和跟踪实验。

视频: Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

科研

免疫与感染

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

中性粒细胞衍生微粒的分离与鉴定功能研究

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.