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><tbody><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>RPMI 1640 1X with L-glutamine and 25 mM HEPES</td><td>Cellgro</td><td>10-041-CV</td><td>&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (Heat inactivated)</td><td>GemCell</td><td>100500</td><td>&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)</td><td>GIBCO</td><td>15140</td><td>&amp;nbsp;</td></tr><tr><td>0.5 M EDTA</td><td>Quality Biological Inc</td><td>351-027-101</td><td>&amp;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 &amp;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 &amp;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>&amp;nbsp;</td></tr><tr><td>Ethyl Alcohol (200 proof)</td><td>The Warner Graham Company</td><td>64-17-5</td><td>&amp;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 &amp;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 &amp;deg;C</td></tr><tr><td>Anti-mouse CD45 (Clone 30-F11)</td><td>eBioscience</td><td>170451-82</td><td>&amp;nbsp;</td></tr><tr><td>Anti-mouse Ly6G (Clone 1A8)</td><td>BD Pharmingen</td><td>551461</td><td>&amp;nbsp;</td></tr><tr><td>Anti-mouse Ly6G (Clone 1A8)</td><td>BD Pharmingen</td><td>560599</td><td>&amp;nbsp;</td></tr><tr><td>Anti-mouse CD11b (Clone M1/70)</td><td>eBioscience</td><td>47-0112-82</td><td>&amp;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>&amp;nbsp;</td></tr><tr><td>Paraformaldehyde Solution, 4% in PBS</td><td>USB Corporation</td><td>19943</td><td>&amp;nbsp;</td></tr><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Table 1. Reagents for isolation, labeling and tracking of adoptively transferred neutrophils.&lt;/strong&gt;</td></tr><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>C57BL/6 mice</td><td>Taconic</td><td></td><td>&amp;nbsp;</td></tr><tr><td>15 ml centrifuge tubes</td><td>Corning</td><td>430053</td><td>&amp;nbsp;</td></tr><tr><td>50 ml centrifuge tubes</td><td>BD Falcon</td><td>352070</td><td>&amp;nbsp;</td></tr><tr><td>25 ml serological pipettes</td><td>Celltreat</td><td>229225B</td><td>&amp;nbsp;</td></tr><tr><td>10 ml serological pipettes</td><td>Celltreat</td><td>229210B</td><td>&amp;nbsp;</td></tr><tr><td>5 ml serological pipettes</td><td>Celltreat</td><td>229205B</td><td>&amp;nbsp;</td></tr><tr><td>Pasteur pipettes (3 ml)</td><td>BD Falcon</td><td>357575</td><td>&amp;nbsp;</td></tr><tr><td>12 ml syringes</td><td>Kendall monoject</td><td>512878</td><td>&amp;nbsp;</td></tr><tr><td>25 G x 5/8 in. Needles (precision glide needles)</td><td>BD</td><td>305122</td><td>&amp;nbsp;</td></tr><tr><td>100 mm cell strainers</td><td>BD Falcon</td><td>352360</td><td>&amp;nbsp;</td></tr><tr><td>Bactericidal Petri dishes</td><td>BD Falcon</td><td>351029</td><td>&amp;nbsp;</td></tr><tr><td>Combitips Plus Biopur</td><td>Eppendorf</td><td>2249608-5</td><td>&amp;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>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Tissue culture hood</td><td>The Baker Company</td><td>SG403</td><td>&amp;nbsp;</td></tr><tr><td>Refrigerated centrifuge</td><td>Thermo Fischer Scientific</td><td>75004521</td><td>&amp;nbsp;</td></tr><tr><td>37 &amp;deg;C shaking water bath</td><td>Thermo Fischer Scientific</td><td>3166721</td><td>&amp;nbsp;</td></tr><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Table 2. List of Materials and Equipment used in this protocol.&lt;/strong&gt;</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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

61.7K 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

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 ...

科研

免疫与感染

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 ...

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.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

新生儿骨髓来源巨噬细胞的吞噬试验评价 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

作者聚焦：开发一种从新生小鼠中分离骨髓来源的巨噬细胞的可重复方法，以促进早期免疫反应

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

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

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此视频中的章节

隔离和小鼠骨髓的静脉注射单核细胞衍生

中性粒细胞衍生微粒的分离与鉴定功能研究

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备