这项工作是由加州大学洛杉矶分校的治疗中心，克罗恩病和结肠炎基金会美国职业发展奖（2691）和国立卫生研究院NIDDK K01（DK084256）的资金就围冠试点和可行性研究的资助。 伯纳德·莱文和Scott厨房加州大学洛杉矶分校艾滋病研究鼠/人嵌合体的核心设施中心的协助下照射骨髓操作。流式细胞仪操作协助加州大学洛杉矶分校矢量的核心设施。

1。在你启动技术注意事项<ol><li>我们建议同时使用C57/BL6野生型小鼠和基因敲除小鼠可以购买相应的CD分子的小鼠实验，因为从各大鼠标厂商。 </li><li>因为它是难以跟踪的野生型小鼠和衍生的基因敲除小鼠骨髓移植后的供体细胞，它是必要使用CD45.1代表野生型小鼠的小鼠。 （CD45.1 C57/SJL WT小鼠杰克逊实验室的股票002014）。 </li><li>大多数鼠标的菌落CD45.2。我们的基因敲除小鼠属于CD45.2的基因型（或者一个CD45.2 WT小鼠来源:杰克逊实验室的股票＃000664）。本协议第7条中提到的，可以使用流式细胞仪检测骨髓和外周血的CD45.1和CD45.2基因型的测定。 </li><li>照射后小鼠免疫力下降。养老鼠在无病原体设施。 </li><li>操作辐照需要特殊安全clearance和培训。为了节省时间，在培训和审批程序，最好是与一个合格的技术人员能够操作辐照实验室。 </li><li>许多机构都流式细胞仪检测实验室，拥有经验丰富的技术人员。为了尽量减少用流式细胞仪操作的问题，最好是经验丰富的技术人员，然后再开始讨论流式细胞仪检测实验计划。 </li><li>小鼠（每组10只小鼠）以这种方式被分配: </li></ol><table border="1"><tbody><tr><td> 组 </td><td></td><td> 到收件人的骨髓捐赠者 </td><td> 治疗 </td></tr><tr><td>一</td><td> BM交流</td><td> CD45.1 WT CD45.2 KO </td><td> DSS结肠炎</td></tr><tr><td>乙</td><td></td><td></td><td>水正常对照</td></tr><tr><td> Ç </td><td> BM交流</td><td> CD45.2 KO CD45.1 WT </td><td> DS性结肠炎</td></tr><tr><td> ð </td><td></td><td></td><td>水正常对照</td></tr><tr><td> Ë </td><td>假</td><td> CD45.1 WT CD45.1 WT </td><td> DSS结肠炎</td></tr><tr><td> F </td><td></td><td></td><td>水正常对照</td></tr><tr><td> ĝ </td><td>假</td><td> CD45.2 KO KO CD45.2 </td><td> DSS结肠炎</td></tr><tr><td> Ĥ </td><td></td><td></td><td>水正常对照</td></tr></tbody></table> *简要说明的实验方案如图1所示。 2。受体小鼠照射<ol><li>小鼠繁殖，并保持在无病原体的设施。将灭菌照射小鼠饼过滤器。将一个鼠标在每个插槽的照射馅饼。将饼进入辐照装置和，确保转盘和饼转向。 </li><li>打开空气泵用于通风。辐照门关闭并锁定。 </li><li>照射的小鼠千拉德（这是相当于10戈瑞）为10分钟左右（根据辐射源和半衰期）。从这个时间点，照射小鼠是免疫能力受损，对感染的抵抗力弱。照射后，采取的馅饼，并将其放置在无菌容器中，动物运输生物安全柜设施。避免暴露小鼠的外部环境，以减少感染的机会。 </li><li>在生物安全柜中，将只小老鼠变成的蒸压鼠标笼，用无菌床上用品（每笼4只。添加新的无菌水与sulfatrim暂停 - 3.12毫升，每100毫升水）。 </li><li>瓶用铝箔包裹的水作为抗生素是光敏感的。摇动混合的sulfatrim解决方案瓶，以及在加载它的笼子前。 </li></ol> 3。捐赠者的骨髓提取<ol><li>与二氧化碳或isoflur的牺牲小鼠ANCE，然后浸泡在1:200的来苏尔溶液。喷洒的小鼠用70％乙醇润湿的皮肤和解剖开放的骨骼。 </li><li>在70％的乙醇消毒浸泡解剖工具。 </li><li>在生物安全柜中进行解剖。使用无菌暴露肱骨，股骨，胫骨和腓骨骨，附着的肌肉和韧带的解剖工具。 </li><li>显示红色的骨髓，剖开两端的骨头。用钳子按住骨头。红骨髓，用冷PBS冲洗3毫升注射器和25G的针头培养皿中。两端的骨头冲​​洗，以产生更多的骨髓细胞。 </li><li>使用6毫升注射器和18G1 / 2针的红骨髓插头的反复抽吸和弹射打破。用50ml Falcon管中，骨髓转移。 </li><li>向下自旋骨髓在转速为1,800 rpm，5分钟，在4℃下涡旋5-10秒，弃上清，重悬沉淀。 </li><li>用无菌水稀释10X红细胞裂解缓冲液。广告ð1X红细胞裂解缓冲液（5毫升，每供体小鼠），旋涡，持续5秒，并再次保持恰好3分钟在室温下。 </li><li> 3分钟后，填写管，用20毫升冷PBS停止裂解和组合。倒入到一个新的50毫升Falcon管中的内容直接通过40μm的细胞过滤网。 </li><li>用5ml的PBS中，并转移到细胞过滤网，以及冲洗原始管。补足至50毫升PBS和组合。 </li></ol> 4。骨髓供体细胞计数<ol><li>加入100μl的台盼蓝到Eppendorf管中骨髓悬浮液和100μl到Eppendorf管中，混合。染色的细胞混合物移液器血细胞计数。 </li><li>生活总骨髓细胞计数=未染色的细胞数在16个正方形×2（由于台盼蓝稀释）×10,000×50毫升Falcon管中的细胞的量的计算方法是</li><li>降速的骨髓细胞在50ml Falcon管中以2,000 rpm在4℃下5分钟</li><li>除去上清液。基于的总细胞计数，将沉淀重悬于用PBS至1×10 8个细胞每毫升</li></ol> 5。输液受体小鼠<ol><li>热烈的受体小鼠热板和下一个变暖的灯。将受体小鼠中限制器。在100-200微升静脉注射1×10 7细胞，每只小鼠。 </li><li>必须注入捐赠者的骨髓照射后4-24小时之间。 </li><li>随时掌握每笼的小鼠注射。第4周在无病原体的动物设施的抗生素处理后的水注入受体小鼠在蒸压笼，保持和让老鼠恢复免疫力。更改笼子里，抗生素处理过的水和食物，每4天，以保持卫生。 </li></ol> 6。结肠炎诱导结肠炎和评价<ol><li>照射和骨髓移植4周后，切换到普通水，无抗生素，并保持2个星期的小鼠，以恢复其正常肠道菌群。 </li><li>骨髓注射6周后，测定初始体重。饮水自由采食诱导结肠炎小鼠给予5％硫酸葡聚糖。一些老鼠经常饮用的水只作为正常对照组。 </li><li> 5天后，提取外周血，转移到肝素涂层管（真空采血管），并保存在冰中。池的血从4只到1管。结合用300μl的细胞染色缓冲液= 600微升300微升血液。血液除以5 Eppendorf管中，每个用100μl。 </li><li>牺牲的老鼠与二氧化碳或异氟醚。评估宏观损伤评分（的详细信息，请参阅视频发布到另一个JOVE）。测量体重变化。用游标卡尺测量肠管长度和厚度。观察大便的质地（硬，软或带血）。确定与Hemooccult大便潜血。解剖结肠组织和固定在福尔马林H＆E染色。 </li><li>解剖1股骨博定义每鼠标，提取骨髓，用冷PBS冲洗骨髓，通过25G针和1毫升注射器。游泳池4骨髓插一管，并保存在冰中。 </li><li>骨髓倒入陪替氏培养皿，剪切骨髓插头用18G注射针和5毫升注射器数次直到没有骨髓插件是本。 </li><li>自旋向下的5分钟1500转，在4°C去除上清，重悬于600μL细胞染色缓冲液。再悬浮的骨髓除以5 Eppendorf管中，每个用100μl。 </li></ol> 7。流式细胞仪检测骨髓移植的质量检验<ol><li>标记每个样品管中的血液或骨髓＃1-5: </li><li>制备抗体混合物在黑暗中为每个各自的管。 </li></ol> ＃1抗体 ＃2 30μLFITC同型对照+ 30μLPE同型对照+ 15微升CD16/32阻塞 ＃3 30μLFITC CD45.1 AB + 15微升CD16/32阻塞 4月30日微升PECD45.2 AB + 15微升CD16/32阻塞 ＃5 30μLFITC CD45.1抗体+ 30微升PE的CD45.2抗体+ 15微升CD16/32阻塞<table border="1"><tbody><tr><td>没有血液或骨髓样本＃1 </td></tr><tr><td>加入5μl的抗体混合物＃2＃2每个血液或骨髓样本</td></tr><tr><td> 3微升的抗体混合物＃3，每个血或骨髓样本＃3 </td></tr><tr><td>每个血液或骨髓样本＃4中加入3μl抗体混合物＃4 </td></tr><tr><td>加入5μl的抗体混合物第5到每一个血液或骨髓样本＃5 </td></tr></tbody></table><ol start="3"><li>查看在黑暗中30分钟，在冰中。 </li><li>每管加入2 ml 1X红细胞裂解液。查看在黑暗中在冰上15分钟。 </li><li>降速的细胞以1,500 rpm，在4℃下5分钟（GH 3.8转子，1500转= 350 XG）除去上清液。重悬沉淀用500μl细胞染色缓冲液在黑暗中，当时的涡简要。 </李&gt; <li>检查CD45.1（代表WT），CD45.2（即KO）的免疫染色的血液和骨髓样本，通过流式细胞仪检测。选择FITC代表WT CD45.1，PE的代表KO CD45.2。 </li><li> FlowJo软件分析流式细胞仪检测结果。计算的比例FITC:市盈率PE:FITC比，以确定是否在血液和骨髓捐助者的基因型是优势基因型。 </li></ol>

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没有利益冲突的声明。

骨髓移植的方法是适用于免疫学研究的结肠炎，感染，癌症，肥胖症和其他疾病的。基因的利益时，需要在两个骨髓来源的和非骨髓来源的细胞和基因的利益被怀疑由细胞介导疾病无论从人口表示这骨髓移植实验。例如，抗菌肽抗菌肽调节急性结肠炎。但是，它是表示在两个上皮细胞和免疫细胞（如巨噬细胞）。然后，我们用骨移植来定义人口的细胞调节小鼠急性肠炎。结肠炎的严重程度显着改变后通过骨髓移植的骨髓抗菌肽基因型的变化。然后，我们可以得出这样的结论骨髓源性细胞中表达抗菌肽起到了重要的作用，在调节急性肠炎的响应DSS。 骨马rrow衍生细胞通常包括红血细胞，白血细胞和血小板。骨髓移植改变这些细胞，但不是别人的基因型。横向来说，这个实验只能在骨髓来源的细胞与非骨髓来源的细胞分化的作用的基因的利益。但是，本实验中，不能进一步定义人口骨髓衍生的所有的红血细胞，白血细胞和血小板的结肠炎来源于骨髓的细胞介导的​​。骨髓衍生细胞迁移到冒号结肠炎的命运是积极和具争议性的研究领域。骨髓移植后的和诱导的小鼠结肠炎，很可能存在一些骨髓衍生细胞淋巴细胞和肌成纤维细胞在冒号5。另一份报告表明，骨髓来源的细胞作为新生血管内皮祖细胞在恢复过程中6。也有evidencË显示骨髓来源的细胞是上皮细胞分化与结肠炎患者7。我们不能排除这种可能性，一些骨髓来源的细胞可以分化典型的免疫细胞以外的细胞，并发挥调节作用结肠炎发展。尽管如此，骨髓来源的单核细胞/巨噬细胞的人口是很重要的保护，防止化学诱导结肠黏膜损伤8,9。本报告是一致的，我们发现，抗菌肽表示，从骨髓衍生细胞调节DSS结肠炎，同时抗菌肽分泌的单核细胞/巨噬细胞4。 另一方面，有很多方法可以跟踪骨髓移植的成功。流式细胞仪分析CD45.1和CD45.2型的，可以提供定量的方法来确定捐赠者的骨髓干细胞来源的细胞在受体小鼠的比例。骨髓细胞s可以分化成多个单元10种。当流式细胞分析是不可行的，这是可能的，使用男性供体小鼠（XY染色体）和女性（XX染色体）受体小鼠11。在XX唯一的女性受体小鼠体内的供体小鼠进行独有的Y染色体。 Y染色体可以通过荧光原位杂交技术11。此外，免疫组织化学CD45.1，CD45.2的和/或在组织中的兴趣基因以可视化的捐赠者的骨髓来源的细胞，可能是必要的。 但CD45流式细胞分析和Y染色体原位杂交程序中通常是这样做后，将小鼠处死。要监视的位置被用来研究慢性疾病，如癌症，而不牺牲小鼠在受体小鼠的供体细胞，它是可能的，使用绿色荧光蛋白的转基因小鼠作为供体小鼠12。因此，捐赠者骨髓行衍生细胞可以在体内受体小鼠的非侵入性的高分辨率光学成像下跟踪瞬态麻醉下，这是可以做到反复。 不是所有的老鼠都成功的骨髓移植13。我们观察到10-20％的小鼠在第2周照射后，由于贫血或感染死亡。因此，应准备比实际需要更多的小鼠在实验开始。例如，你应该准备组10只小鼠在实验开始的，如果你希望结肠炎实验结束时每组8只小鼠。此外，请确保您删除的死老鼠，尽快。免疫重建的速度相关的编号，造血干细胞在骨髓注入受体小鼠14。因此，至关重要的是有足够数量的活的骨髓细胞（1×10 7细胞每只小鼠）注入受体小鼠成功骨髓TR的ansplantation。 收件人照射后小鼠免疫力下降。供体小鼠解剖和应该做骨髓编写的程序，在相同的标准细胞培养实验。使用无菌工具和容器，应适用的无菌处理技术。在整个所有的实验中，PBS包含1％青霉素 - 链霉素和10 U / ml的肝素应骨髓的处理过程中使用。所有的试剂，细胞培养级。可以发现更多的技术讨论，参考文献13。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>细胞染色缓冲液</td><td> Biolegend </td><td> ＃420201 </td></tr><tr><td> 10X红细胞裂解缓冲液</td><td> Biolegend </td><td> ＃420301 </td></tr><tr><td> FITC同型对照鼠</td><td> Biolegend </td><td> ＃400207 </td></tr><tr><td> PE同型对照鼠</td><td> Biolegend </td><td> ＃400211 </td></tr><tr><td> PE抗鼠CD45.2 </td><td> Biolegend </td><td> ＃109807 </td></tr><tr><td> FITC标记的抗小鼠CD45.1 </td><td> Biolegend </td><td> ＃110705 </td></tr><tr><td>抗小鼠CD16/32阻塞</td><td> Biolegend </td><td> ＃101301 </td></tr><tr><td> 40微米的细胞过滤器</td><td> Fisherbrand </td><td> ＃22363547 </td></tr><tr><td> PBS 1X </td><tD&gt; MP biomedicals </td><td> ＃1860454 </td></tr><tr><td>肝素</td><td> Fisherbrand </td><td> ＃BP2425 </td></tr><tr><td> Sulfatrim（SMZ） </td><td> Qualitest </td><td> NC9242720（费舍尔） </td></tr><tr><td>来苏儿IC </td><td> Andwin科学</td><td> NC9745686（费舍尔） </td></tr><tr><td> Vaccutainer </td><td> BD </td><td> ＃8000813 </td></tr><tr><td>鼠标阻挡</td><td>布伦特里科学</td><td> ＃TV-150 </td></tr><tr><td>照射器</td><td> JL牧羊犬和联营公司</td><td> Mark我68A </td></tr><tr><td>流式细胞仪</td><td> BD </td><td> BD FACSCanto II </td></tr><tr><td>流式细胞仪检测管</td><td>鹰</td><td> ＃352052 </td></tr><tr><td>数显卡尺</td><td> Fisherbrand </td><td> 14-648-17 </td></tr><tr><td>大便潜血信息和通信技术</td><td> Beckman Coulter公司</td><td> G0328QW </td></tR&gt; </tbody></table>

differentiating functional roles of gene expression from immune and non-immune cells in mouse colitis by bone marrow transplantation

要了解结肠炎的发展中的一个基因的作用，我们比较了野生型小鼠的反应和兴趣基因缺陷的基因敲除小鼠结肠炎。如果该基因的利益是表示在两个骨髓来源的细胞和非骨髓来源的细胞的主机，但是，它是可能的感兴趣的基因的骨髓来源的细胞和非骨髓分化的作用衍生骨髓细胞​​移植技术。要改变的骨髓细胞基因型的小鼠，原来的受体小鼠的骨髓被破坏的照射，然后更换新的捐赠者的骨髓不同基因型。当野生型小鼠供体骨髓移植到基因敲除小鼠中，我们可以产生与野生型的骨髓来源的细胞中的基因表达的基因敲除小鼠。或者，基因敲除小鼠时，供体骨髓移植到无利益基因表达的野生型受体小鼠，野生型小鼠从骨髓衍生细胞。然而，骨髓移植可能不是100％完成。因此，我们利用集群的分化（CD）分子（CD45.1和CD45.2）作为供体和受体细胞的标记，跟踪捐赠者的骨髓来源细胞在受体小鼠的骨髓移植成功的比例。 CD45.1 CD45.2基因型的基因型和基因敲除小鼠与野生型小鼠。受体小鼠的照射后，供体骨髓细胞注入受体小鼠不同基因型。当新的骨髓再生接管其免疫，将小鼠用化学剂（硫酸葡聚糖钠，DSS 5％）的挑战，以诱发大肠炎。在这里，我们还表明，该方法诱导结肠炎的小鼠的骨髓来源的细胞从表达感兴趣的基因的作用的评价。如果该基因的利益从骨来源的细胞的疾病的发展中起着重要的作用（如大肠杆菌炎），骨髓移植的受体小鼠的表型可以显着改变。结肠炎实验结束时，骨髓来源的血液和骨髓中的细胞进行标记与抗CD45.1和CD45.2和其定量的存在比例可用于骨髓移植的成功，通过流式细胞术评价。应显示成功的骨髓移植供体基因型在长期的CD分子标记对收件人基因型的受体小鼠的骨髓和血液中的绝大多数。

如果该基因的利益结肠炎的开发过程中，起到了重要的作用的免疫细胞，通过骨髓移植（WT KO KO，以WT）小鼠骨髓不同基因型，DSS结肠炎应该有一个改变的响应。用于确定的结肠炎的严重程度的最重要的参数之一是结肠组织的H＆E染色。结肠组织结构的变化和炎症的迹象，H＆E组织学评分系统，可以定量评估。 H＆E组织学得分DSS结肠炎模型的标准可以在这里找到2。另外，也可诱发化学诱导结肠炎三硝基苯磺酸（TNBS）。 TNBS结肠炎诱导和H＆E组织学评分标准的方法，可以发现在之前发表的3。组织学评分两组之间的差异可以分析学生的t检验。 小鼠可能有显着假体骨髓移植（WT WT或KO KO）的小鼠相比，改善或加重结肠炎。如果该基因的利益起到了重要的作用结肠炎通过骨髓源性细胞，小鼠骨髓交换反应显着不同的假体骨髓移植的小鼠。 DSS结肠炎模型，组织学变化进行评估，通过组织学评分（ 见图4）。组织学评分显着变化可以指示由骨髓来源的细胞中的兴趣基因介导的结肠炎的发展。例如，抗菌肽是一种抗微生物和抗炎肽基因（在我们的例子中的兴趣基因）4。如果没有骨髓移植，抗菌肽基因敲除小鼠比野生型小鼠在响应DSS开发更糟糕的结肠炎。输血导致野生型（WT）的抗菌肽基因敲除（KO）小鼠骨髓改进后的结肠炎transfusi从抗菌肽基因敲除（KO）小鼠，以野生型（WT）小鼠骨髓导致恶化结肠炎DSS（ 图2）时，接触到。 为了验证利益的基因表达，基因表达的骨髓供体野生型基因的利益（ 如抗菌肽）在结肠（或其他）的的抗菌肽缺乏淘汰赛受体小鼠后骨组织检测骨髓移植。也很想知道，是否减少基因敲除小鼠的骨髓捐赠后表达的基因在野生型受体小鼠的利益。 成功植入捐赠者的骨髓为代表的收件人的CD同型中的受体小鼠外周血细胞和骨髓供体CD同种以上的优势比。骨髓示出最好的标记在流式细胞术中的血液未染色的细胞（ 图2和图3）有一个特定的CD45.1和CD45.2。 图1。骨髓移植的实验方案。 <img alt="图2" fo:content-width="5.6in" fo:src="/files/ftp_upload/4208/4208fig2highres.jpg" src="/files/ftp_upload/4208/4208fig2.jpg" /> 图2。骨髓移植后受体小鼠的骨髓的流式细胞仪数据的 Y轴表示FITC-标记的CD45.1信号和X轴显示了PE-标记的CD45.2信号。骨移植成功的定义是在受体小鼠骨髓和血液中的CD45.1和CD45.2基因型的变化。 <a href="http://www.jove.com/files/ftp_upload/4208/4208fig2large.jpg" target="_blank">点击此处查看大图</a> 。 图3。流式细胞术受体小鼠骨髓移植后的血液数据的 Y轴表示，FITC标记CD45.1信号和X轴显示了PE-标记的CD45.2信号。骨移植成功的定义是在受体小鼠骨髓和血液中的CD45.1和CD45.2基因型的变化。 <a href="http://www.jove.com/files/ftp_upload/4208/4208fig3large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图4" fo:content-width="4.5in" fo:src="/files/ftp_upload/4208/4208fig4highres.jpg" src="/files/ftp_upload/4208/4208fig4.jpg" /> 图4。结肠炎的小鼠骨髓移植后的评价。（A）H＆E影像冒号与n正式的组织学和DSS结肠炎。 （B）组织学分数。成功地诱导DSS结肠炎组织学评分显着增加，可以确认的第5天的DSS处理。骨髓不同基因型的交流后，组织学评分应该改变显着。这表明由该基因的感兴趣的骨髓来源的细胞中的表达的过程中改变结肠炎。数据被表示为平均值±标准误差的装置。

Watch this Scientific Journal Video about Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation at JoVE.com

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

骨髓移植提供了一种方法来改变的骨髓衍生细胞的基因型。如果感兴趣的基因的表达在两个骨髓来源的细胞和非骨髓来源的细胞，骨髓移植可以改变到一个不同的基因型的骨髓来源的细胞，不改变非骨髓来源的细胞的基因型。

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 ...

differentiating-functional-roles-gene-expression-from-immune-non

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.4K 次观看.  The University of California Los Angeles, Los Angeles. 骨髓移植提供了一种方法来改变的骨髓衍生细胞的基因型。如果感兴趣的基因的表达在两个骨髓来源的细胞和非骨髓来源的细胞，骨髓移植可以改变到一个不同的基因型的骨髓来源的细胞，不改变非骨髓来源的细胞的基因型。

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

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

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

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 ...

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 ...

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 ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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 ...

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 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...