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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The authors have no disclosures.

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...

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 border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:301px;">Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:343px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Mouse IgE FITC</td>
			<td>BD Pharmingen</td>
			<td>553415</td>
			<td>clone R35-72</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgG1 PE</td>
			<td>BD Pharmingen</td>
			<td>550083</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Methanol</td>
			<td>BDH</td>
			<td>BDH1135-4LP</td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-4052-82</td>
			<td>clone RA3-6B2</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">anti-Mouse/Rat CD40</td>
			<td>eBioscience</td>
			<td>16-0402-85</td>
			<td>clone HM40-3</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MEM-NEAA (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Phosphate Buffered Saline (10X)</td>
			<td>Lifetechnologies (Corning)</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;">MACS CD45R (B220) microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-501</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MACS purification column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">IL-4</td>
			<td>PeproTech</td>
			<td>214-14</td>
			<td>Reconstitute in water or 0.1% BSA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Formalin Solution, Neutral Buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Trypsin-EDTA Solution (10X)</td>
			<td>Sigma Aldrich</td>
			<td>T4174-100mL</td>
			<td>5.0g Trypsin, 2.0g EDTA&#376;4Na per Liter of 0.9% NaCl</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100mL</td>
			<td>10,000U Penicillin; 10 mg/mL Streptomycin (100X)</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">L-Glutamine 200mM</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">2-mercaptoethanol (99%)</td>
			<td>Sigma Aldrich</td>
			<td>M6250-100mL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Red cell lysis buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgM RPE/cy7</td>
			<td>SouthernBiotech</td>
			<td>1140-17</td>
			<td>clone 1B4B1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HyClone Fetal Calf Serum</td>
			<td>Thermo</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;">B cell Stimulation media</td>
			<td></td>
			<td></td>
			<td style="width:343px;">B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,&#160; 1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &#181;g/ml), Beta-mercaptoethanol (7 &#181;L/L), IL-4 (20 ng/mL), and anti-CD40 (1 &#181;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?...

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

Research

JoVE Journal

Immunology and Infection

10.8K 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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

纯净小鼠的B细胞的诱导和评估类开关重组

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

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 ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

检测真正的IgE的表达小鼠B​​系细胞

检测真正的IgE的表达小鼠B系细胞