Name: detection of true ige-expressing mouse b lineage cells
Uploaded: 2014-12-01T14:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

DRW是由美国国立卫生研究院资助AI089972和AI113217，并通过粘膜免疫学研究团队的支持，并拥有一个事业奖从宝来惠康基金医科大学的科学家。

注:此处描述的所有实验都按照实验动物管理和使用委员会（IACUC）的指导方针并经动物研究儿童医院（ARCH），马萨诸塞州波士顿。 1.试剂的配制<ol><li> 1000倍的IL-4库存:稀释的IL-4细胞因子，以20微克/毫升的H 2 O或0.1％牛血清白蛋白（BSA）。在200μl等份分配到1.5ml微量管中并储存在-20℃。 </li><li> 1000倍的抗CD40:从制造商获得，在1.0毫克/毫升，保存于4℃。 </li><li> B细胞刺激介质:向425毫升RPMI-1640培养基中，添加75毫升刚解冻的胎牛血清（FCS），10毫升的1.0M HEPES缓冲液，加入5ml L-谷氨酰胺的库存，将5ml青霉素:链霉素库存，5毫升最小必需培养基非必需氨基酸（MEM-NEAA），和3.5微升2-巯基乙醇。媒体保持在4℃长达一个星期。添加IL-4（20毫微克/毫升）和抗CD40即将使用之前（1.0微克/毫升）只对所需的介质的体积。 </li><li> 2％FCS-FACS缓冲:加入10 mL FCS来490毫升1X PBS的。保持在4℃。 </li><li> 2×胰蛋白酶-EDTA溶液稀释:将5ml 10×胰蛋白酶-EDTA原液（5.0克胰蛋白酶，每升2.0克EDTA）中于20毫升1×PBS中。商店胰蛋白酶-EDTA在2X工作等分试样在4℃下。允许温热至室温后使用。 </li><li>胎牛血清:保持解冻的FCS在4℃至两周。细胞质染色过程前放置在冰上15分钟。 </li><li>中性缓冲福尔马林:准备10％的福尔马林溶液（3.7％多聚甲醛）。甲醛是有毒的，因此处理它在实验室通风柜，而穿着适当的个人防护装备。福尔马林储存在室温下的化学品储存柜。 </li><li>甲醇:存放无水甲醇（100％），在-20℃直到细胞已准备好进行透化。需要注意的是甲醇是易燃的，并且被存储在一个适当的冰柜。 </li></ol> 2。净化和B细胞的激活<ol><li>从新鲜收获小鼠脾脏，通过一个70微米的细胞滤网用3毫升注射器的柱塞轻轻按压器官制备单细胞悬浮液。在一个15毫升锥形离心管中，细胞收集在10ml的1×PBS和离心机在300×g离心5分钟，在4℃下。 </li><li>倒出1毫升红细胞溶解缓冲液上清，重悬沉淀5分钟。与13.5毫升1×PBS中，并中和离心机在300×g离心5分钟。 </li><li> （可选的）磁分离使用抗CD45R（B220）的B细胞 - 标记珠，以下，根据制造商的说明将MACS纯化方案。 </li><li>刺激纯化的B细胞以1.0×10 6个细胞/在含IL-4（20毫微克/毫升）和抗CD40（1.0微克/毫升）5天温热B细胞刺激媒体毫升，在37℃。开始用8ml培养的分成2孔6孔培养板（4每毫升）的。 </li><li>检查文化前进重新每日在1.0×10 6个细胞/ ml来维持。通过分割成其他井和与含有新鲜的IL-4和抗CD40的B细胞的刺激介质稀释该培养调节浓度。 </li></ol> 3.胰蛋白酶消化，固定和通透<ol><li>胰蛋白酶<ol><li>收集到1.0培养细胞×10 7在15ml锥形管中，离心机在300×g离心5分钟，倾倒出上清液。 </li><li>用10ml的PBS洗涤，以从细胞收集培养基除去残留的蛋白质。重复离心步骤，倒出上清液。 </li><li>倾析（约100微升）后悬浮在剩余体积的细胞沉淀。 </li><li>小心添加800微升室温0.1％（2×）胰蛋白酶-EDTA细胞中。分配慢慢在45°角下的锥形管的一侧。 </li><li>关闭该管并且通过倾斜管轻轻混匀，以便将液体沿着所述管5-6的时间长度运行秒。一粘性的，白色沉淀可以形成在溶液中。限制胰酶消化时间降至最小下。 </li><li>淬火用1ml冷的FCS的胰蛋白酶反应。立即加入10毫升冷的PBS稀释的FCS中。 </li><li>离心机在300×g离心5分钟，在4℃，然后倒出上清液并返回管到冰上。 </li></ol></li><li>固定术<ol><li>倾析后悬浮细胞中的残留量。如果有必要，把音量高达100微升冷PBS。 </li><li>在通风橱中，加入800微升10％中性缓冲福尔马林溶液至细胞和吸管上下。管将温暖由于加入福尔马林。孵育在37℃下进行10分钟。 </li></ol></li><li>通透性<ol><li>成立了台式旋涡混合器在轻轻摇动的速度。 </li><li>绘制9毫升的冷（-20℃）甲醇到10ml血清吸管。 </li><li>持含有细胞在涡流混合器的管，轻轻启动沙金克细胞。 </li><li>加入冷的甲醇到管使其滴落到细胞而管仍然振摇使甲醇被连续混合。后的前2毫升甲醇中的分配速度可以提高到一个缓慢流。 </li><li>让管坐于冰上30分钟，以完成该透。或者，所述细胞可以储存在-20℃下长达一个月。 </li></ol></li></ol> 4.荧光染色的IgG1和IgE的类别转换<ol><li>在300×g离心5分钟，离心样品，倒出甲醇溶液。白色沉淀物可能沉淀的细胞。 </li><li>加入10毫升室温的1×PBS和离心机在300×g离心5分钟以洗涤细胞。重复一次。注意:清洗用室温PBS颗粒溶解多余的沉淀物可能形成步4.1后。 </li><li>用10毫升2％FCS的FACS缓冲液进行一个额外的洗涤。 </li><li>分发样品到96孔圆底板并离心，在300×g离心5分钟。滗上清液并把该板在冰上。 </li><li>染色在60微升的2％FCS的FACS缓冲液，每孔用下列抗体稀释液:抗IgM的RPE / Cy7的，1:200，反的IgG1 PE，1:600，抗IgE FITC，1:100，反CD45R（B220）PerCP-经Cy5.5，1:200 </li><li> 5分钟后染色，用150微升2％FCS的FACS缓冲液，并离心洗涤平板5分钟，在300×g下。 </li><li>重悬在400微升2％FCS的FACS缓冲液和吸管通过40微米尼龙细胞过滤到5ml的FACS管。 </li></ol> 5.使用以下门控策略: <ol><li>在爆破淋巴细胞群体位于FSC / SSC 图 （ 图1）隔离CD45R（B220）阳性细胞。 </li><li>从B220阳性细胞，用IgE和IgG1的剧情揭示相应的人群。 </li></ol>

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小鼠脾B细胞在培养用抗CD40和IL-4的刺激将模拟T辅助2型（T H 2）的相互作用，激励类别转换为IgG1和IgE 5。 B细胞可用于CSR在总脾细胞12或作为纯化的脾B细胞11的情况下被激活。在协议中（步骤2.3）所指出的，B细胞富集是可选的，并且是在实验者的判断，以确定它是否将是有益的。使用CD45R（B220）B细胞的正磁分离标记的珠子在这里使用。分离后，建议通过FACS来检查细胞的纯度，以使B细胞浓度可以调整到1.0×10 6个细胞/ ml进行适当的细胞因子和抗体的刺激。还应当指出的是，IL-4蛋白，其已经经历冻结/解冻循环可能已降低的活性。 细胞之前固定和透化的胰蛋白酶允许ACCUR通过流式细胞仪吃类别转换的测量（ 图2）。适当的胰蛋白酶消化细胞，是该过程的成功应用是至关重要的。作为FCS的能抑制胰蛋白酶，一个1×PBS中洗涤之前进行的胰蛋白酶消化步骤中进行。我们发现了30秒的温育在室温下足以完全消化。较长时间可以显着影响细胞活力。 在分析之前，有必要多次用PBS洗细胞以除去痕量的甲醇。甲醇残留可能会改变抗体结合13，可以从沉淀的蛋白质FCS下游流式细胞仪分析14使用。在这方面，其它透方法（ 即皂苷）可以是另一种使用甲醇。甲醇是溶解膜脂质，而洗涤剂破坏膜的完整性15的有机溶剂。一个好处甲醇通透的扩展存储能力的固定的细胞在-20℃下14。 有一些限制使用此方法时，要牢记。因为胰蛋白酶切割蛋白的残基，部分的FACS污渍可能出现的治疗后的不同，或可能会失去结合共靶向表位的能力。此外，胰蛋白酶处理后的细胞数的损失可以发生。然而，这不会出现影响的IgE的CSR的测量的准确度，因为我们已发现该方法是明确地比得上从测量个体杂交瘤克隆11的抗体的分泌物收集类别转换的数据。 其他方法已被报道用于检测产生IgE的小鼠的B细胞。一种方法是使用酸洗过程删除受体结合的IgE之前，染色膜结合的IgE对IgE表达细胞16 - 18。另一种方法使用单克隆抗IgE抗体细胞permeabiliz之前阻止所有表面的IgEATION和染色的IgE的表达细胞19细胞内的IgE。此外，最近的一项研究异形的八色FACS面板，以确定在人类IgE的交换的B细胞群体通过排除的IgM +，IgD的+的IgG +和IgA + B细胞亚群20。相比于这些方法，此处描述的方法避免了使用变性酸洗涤的，过量的抗体是没有必要的。 一个好处这个程序是，它并没有规定具体的单克隆抗体CSR的测量IgE水平。替代抗IgE单克隆抗体已经显示出仅检测内源性的IgE分子。的杂交瘤克隆R1E4产生特定仅对内源性表面分子大鼠抗小鼠单克隆IgE的抗体，而不是嗜细胞的IgE结合于CD23 21,22，但并不是市售的。上述协议是方便和经济的，因为它使用普通实验室reageNTS。这里所用的可能的抗IgE抗体结合受体结合的IgE在一个站点到胰蛋白酶介导的切割敏感的，因为IgE的信号由于嗜细胞的IgE治疗后减少（ 图2）。然而研究人员在执行这个协议可以自由从一系列的实验，商用克隆选择。此外，使用胰蛋白酶除去细胞表面蛋白的概念可以扩展到其它细胞标记物通过FACS分析检测。

在免疫球蛋白重链（IGH）类开关重组（CSR）的小鼠和人类， 室内运动场μ恒定区外显子（Cμ）被删除并通过几套下游恒定区外显子的一个（C H S）（ 如更换Cγ， Cε和Cα），导致从生产的IgM的一个开关来产生其它的Ig类（ 例如 IgG抗体，IgE或IgA的）的。 CSR发生内开关（S）的区域，这是位于5&#39;至每一组C H S 1 1-10 kb的序列。激活诱导胞嘧啶核苷脱氨酶（AID）酶通过胞苷脱氨活动启动CSR。 IgE的是过敏性疾病2的关键调解人和IgE水平是如何产生和调控可能会打开大门，新的治疗方法，以过敏性疾病的认识。在小鼠和人类，IgE的是最严格的监管免疫球蛋白同型。是的IgE的水平数千恬正常检测ES小于其他的IgH同种型3，但可以高度升高的疾病状态4。然而，CSR至IgE的是不完全的了解。利用体外激活的B细胞的IL-4与两种抗CD40和LPS结合，诱导的CSR既IgG1和IgE的5。活化的B细胞表达低亲和性IgE受体FcεRII/ CD23 2,6，其结合可溶性IgE的分泌在培养后的CSR。因此，当用流式细胞仪分析，B细胞与受体结合的IgE染色类似于B细胞内源性表达的IgE 7。同时，已知小鼠的B细胞表达低亲和力的IgG受体FcγRIIB1（CD32）8，根据我们的经验它不会出现干扰类别转换为IgG1的测定。然而，B细胞活化培养后测量的IgE切换时，非特异性表面结合的IgE可能掩盖了分析。与普通的染色方法，非IgE表达细胞染色阳性的IgE。 这里所概述的是，已被利用来从小鼠9进行的CSR测定法和检测真正的IgE表达的B细胞的策略- 11。激活B细胞与胰蛋白酶治疗，常见的实验室试剂消化蛋白质，同时删除cytophylic和膜结合表面的IgE。随后的通透和染色质的IgE从而揭示了真正的产生IgE的细胞。

<table><tbody><tr><td>Anti-Mouse IgE FITC</td><td>BD Pharmingen</td><td>553415</td><td>clone R35-72</td></tr><tr><td>Rat Anti-Mouse IgG1 PE</td><td>BD Pharmingen</td><td>550083</td><td>clone A85-1</td></tr><tr><td>Methanol</td><td>BDH</td><td>BDH1135-4LP</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>Falcon Cell Strainer 40 &amp;micro;m Nylon</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Falcon Cell Strainer 70 &amp;micro;m Nylon</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td><td>eBioscience</td><td>45-4052-82</td><td>clone RA3-6B2</td></tr><tr><td>anti-Mouse/Rat CD40</td><td>eBioscience</td><td>16-0402-85</td><td>clone HM40-3</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>MEM-NEAA (100x)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Phosphate Buffered Saline (10x)</td><td>Lifetechnologies (Corning)</td><td>46-013-CM</td><td></td></tr><tr><td>MACS CD45R (B220) microbeads&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-049-501</td><td></td></tr><tr><td>MACS purification column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>IL-4</td><td>PeproTech</td><td>214-14</td><td>Reconstitute in water or 0.1% BSA</td></tr><tr><td>Formalin Solution, Neutral Buffered, 10%</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma Aldrich</td><td>T4174-100 ml</td><td>5.0 g Trypsin, 2.0 g EDTA&amp;#8226;4 Na per Liter of 0.9% NaCl</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P0781-100 ml</td><td>10,000 U Penicillin; 10 mg/ml Streptomycin (100x)</td></tr><tr><td>L-Glutamine 200 mM</td><td>Sigma Aldrich</td><td>G7513</td><td></td></tr><tr><td>2-mercaptoethanol (99%)</td><td>Sigma Aldrich</td><td>M6250-100 ml</td><td></td></tr><tr><td>Red cell lysis buffer</td><td>Sigma Aldrich</td><td>R7757</td><td></td></tr><tr><td>Rat Anti-Mouse IgM RPE/Cy7</td><td>SouthernBiotech</td><td>1140-17</td><td>clone 1B4B1</td></tr><tr><td>HyClone Fetal Calf Serum</td><td>Thermo</td><td>SH30910.03</td><td></td></tr><tr><td>B cell Stimulation media</td><td></td><td></td><td>B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1x MEM-NEAA, 2.0 mM L-glutamine,&amp;nbsp; 1x Penicillin-Streptomycin (Penicillin: 100 U/ml, Streptomycin: 100 &amp;micro;g/ml), Beta-mercaptoethanol (7 &amp;micro;L/L), IL-4 (20 ng/ml), and anti-CD40 (1 &amp;micro;g/ml)</td></tr></tbody></table>

detection of true ige-expressing mouse b lineage cells

B淋巴细胞免疫球蛋白重链（IgH基因）类别转换重组（CSR）是一种方法，其中最初表达IgM的切换到其他的IgH同种型，如IgA，IgE和IgG抗体。的IgH CSR的体外测定是用于若干生物过程包括从DNA重组和修复的分子和细胞免疫学方面的研究的重要方法， 在体外的CSR测定涉及在活化的B细胞的流式细胞仪测量表面Ig的表达。而IgA和IgG亚类的测量是简单的，IgE介导的通过该方法测定是有问题的，由于可溶性IgE结合到FcεRII/ CD23表达活化的B细胞的表面上。在这里，我们描述的产生IgE的，在文化都发生了CSR小鼠B细胞精确测量一个独特的程序。该方法是基于对在细胞表面的IgE CD23复合物的胰蛋白酶介导的裂解，允许检测由CY产生IgE的B谱系细胞toplasmic染色。这个程序提供了企业社会责任的流式细胞仪分析，IgE的一个方便的解决方案。

这个程序已经成功实施，研究企业社会责任，以IgE的小鼠B细胞。为了证明的效率的测量的CSR，我们刺激的小鼠脾B细胞与抗CD40和IL-4如先前所描述的11。刺激后五天，收集细胞并用上述与荧光标记的IgM，的IgG1，IgE和B220（CD45R）抗体染色的协议处理。门控的爆破淋巴细胞（ 图1），的IgE +细胞的明确的人口不能干净地鉴别未经胰蛋白酶处理（ 图2a）。然而，之前的固定和透化细胞的胰蛋白酶处理使所述的IgE生成细胞（ 图2b）的分离由于除去表面结合的IgE。 <img alt="图1" src="/files/ftp_upload/52264/52264fig1highres.jpg" /> 图1:FORWARD与小鼠脾B细胞在5天后刺激在培养中与IL-4和抗CD40侧向散射FACS曲线。爆破淋巴细胞弥补所获取的总的事件的63％。 <img alt="图2" src="/files/ftp_upload/52264/52264fig2highres.jpg" /> 图2:胰蛋白酶对IgE和IgG1的在活化的B细胞的细胞质染色的影响（A，B）FACS图（上图）和直方图（下）选通从129 / B6混合背景的小鼠中活化培养后爆破脾B细胞5天用抗CD40和IL-4。固定和甲醇透单独（a）和与添加胰蛋白酶的（b）示出。

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

Detection of True IgE-expressing Mouse B Lineage Cells

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

detection-of-true-ige-expressing-mouse-b-lineage-cells

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

10.9K 次观看.  Brigham and Women's Hospital and Harvard Medical School. In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

视频: Detection of True IgE-expressing Mouse B Lineage Cells

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

可行的小鼠IL - 17分泌性T细胞的检测和隔离

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

科研

生物学

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

免疫与感染

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

李斯特菌单细胞基因感染后小鼠脾脏原位干扰素伽马生产的成像

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

CXCR4在Murine B-1a细胞上的逆转录病毒过度表达和靶向B-1a细胞向骨髓和IgM生产转移的采用转移

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

医学

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

检测真正的IgE的表达小鼠B系细胞

摘要

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此视频中的章节

可行的小鼠IL - 17分泌性T细胞的检测和隔离

IL-22表达淋巴细胞使用记者小鼠的可视化

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

李斯特菌单细胞基因感染后小鼠脾脏原位干扰素伽马生产的成像

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

CXCR4在Murine B-1a细胞上的逆转录病毒过度表达和靶向B-1a细胞向骨髓和IgM生产转移的采用转移

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

检测真正的IgE的表达小鼠B​​系细胞

摘要

探索更多视频

此视频中的章节

可行的小鼠IL - 17分泌性T细胞的检测和隔离

IL-22表达淋巴细胞使用记者小鼠的可视化

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

李斯特菌单细胞基因感染后小鼠脾脏原位干扰素伽马生产的成像

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

CXCR4在Murine B-1a细胞上的逆转录病毒过度表达和靶向B-1a细胞向骨髓和IgM生产转移的采用转移

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

检测真正的IgE的表达小鼠B系细胞