搜索

Enrichment of Cells of Interest from Heterogeneous Murine Cells with BioMag Goat Anti-rat IgG
用BioMag羊抗大鼠抗体从鼠细胞中富集目的细胞   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

BioMag Goat Anti-Rat IgG is a standard BioMag particle coated with polyclonal goat anti-rat IgG antibodies and is highly suited for use in cell sorting methods where a rat IgG antibody is used as a primary antibody. BioMag Goat Anti-Rat IgG can be used to separate the cells of interest from a heterogeneous cell population using negative selection. BioMag Goat Anti-Rat IgG can also be used as a secondary antibody in enzyme immunoassays and radioassays that utilize a rat IgG primary monoclonal antibody.
The efficiency of enrichment by negative selection depends on antigen availability and the total cell population. The author has intensive experience about enrichment of mouse CD4CD8 double negative (DN) thymocytes from total thymocytes which contains only about 2% DN population. Usually the DN population occupies more than 80% of total thymocyte after one round of enrichment. However the depletion efficiency is much lower for bone marrow cells.

Materials and Reagents

  1. Mouse total thymocytes
  2. BioMag goat anti-rat IgG (QIAGEN, 50 ml, catalog number: 310104 ) (QIAGEN, 500 ml catalog number: 310107 ) (1 mg/ml)
  3. Rat anti-mouse CD4 antibody (BD Biosciences/BD Pharmingen™, catalog number: 553727 )
  4. Rat anti-mouse CD8 antibody (BD Biosciences/BD Pharmingen™, catalog number: 553027 )
  5. BioMag suspension
  6. Thymocytes
    Note: Whether add anti-CD4 antibody to do depletion or cell sorting is controversial because some researcher claimed that a minor subset of DN population, DN1, expresses low level of CD4.

Equipment

  1. Imagnet magnetic separator (BD Biosciences/BD Pharmingen™, catalog number: 552311 , Batch, catalog number: 0000038241 )
    Note: Qiagen also offers magnetic separator suitable for different containers such as single-tube (catalog number: 36910 ), 12-tube (catalog number: 36912 ), 15 ml/50 ml tube (catalog number: 36935 ), and flask (catalog number: 36937 ) as well.

Procedure

  1. Calculation
    1. For optimal results, use the most diluted cellular suspension possible. Typically, total cells are prepared at 2 x 107 total cells/ ml medium. Since any given cell source will have unique purification requirements, it is recommended to determine the optimal condition by individual user.
    2. QIAGEN suggests using 10-50 magnetic particles per cell (total cell population). However it has been proved that as few as 2-3 magnetic particles per total thymocytes works very efficiently. 1 mg/ml BioMag suspension contains 1 x 108 magnetic particles per milligram, which is equivalent to 1 x 108 magnetic particles per milliliter. Therefore, the volume of BioMag suspension required is 2.0-3.0 ml per 108 total thymocytes.

  2. Prepare cell suspension
    1. Adjust the concentration at 2 x 107 total cells/ml sterile culture medium.

  3. Primary antibody binds to cell surface antigen
    1. Add the appropriate amount of primary antibody to the cells and incubate for 30 min at 4 °C or on ice. In order to maximize the binding between the antigens and antibodies, invert the tube contains the cells and primary antibodies several times every 5 min. For optimal results, individual researcher needs to optimize the amount of primary antibody.

  4. Wash BioMag particles
    1. During the procedure of binding, wash required amount of BioMag particles 2-3 times in appropriate sterile culture medium or buffer. First, suspend Biomag particles in original buffer which contains sodium azide, and use a magnetic separator (>20 megaoersted) to pull the magnetic particles to the side of the tube. Suck the original buffer carefully to removes the sodium azide preservative. Separation should be performed for 2-3 min using a magnetic separator.
    2. Repeat the procedure with low protein (5% FCS) buffers are recommended to reduce nonspecific binding.
      Important: Do not centrifuge the BioMag suspension during wash steps. Centrifugation results in extensive aggregation and loss of binding activity.

  5. Remove unbound primary antibody from the cells
    1. Centrifuge at 200-300 x g for 5 min and suck the supernatant. Resuspend the cells in sterile medium and centrifuge at 200-300 x g for 5-10 min to wash the cells. Repeat wash step 1-2 times, using 1 ml sterile medium for each wash.

  6. BioMag particles bind to primary antibody
    1. Resuspend cells in appropriate volume of sterile medium and add the washed BioMag particles prepared in step 3. The final volume of sterile medium and BioMag particles should keep the cells at 2 x 107/ml. Incubate at 4 °C for 15 min. Swirl reaction vessel occasionally, or place eppendorf tubes on a rotating wheel during incubation.
    Notes:
    1. For optimal results, total volume should be ≥ 1ml (including BioMag suspension and cell volume). Where volumes <1 ml are used, additional medium or buffer should be added to a final volume of 1 ml.
    2. Longer incubation is not recommended as magnetic particles may detach from the target cells as a result of cell surface changes over time.
    3. Room temperature (15-25 °C) or 37 °C is optimal for some cell types and QIAGEN.
    4. Recommends that the optimal cell sorting conditions be individually determined.

  7. Separate target cells from other unwanted cells
    1. Apply vessel to a magnetic separator for 10 min at 4 °C. Once separation is complete, carefully remove the supernatant without disturbing the pellet to another tube, the supernatant contains target cells after negative selection.
    Notes:
    1. A clear supernatant indicates that the separation is complete.
    2. Separation must be performed with the vessel held vertically so that the pellet forms on the side of the flask or tube. This ensures unselected cells do not contaminate the magnetic pellet.
    3. For optimal results, repeats step 7 once. The author strongly suggests repeating step 7 once with at least 15 min of separation because any contaminated magnetic particle will interfere with the following experiment especially cell sorting.

  8. Count target cells
    1. Centrifuge the supernatant at 200-300 x g for 5 min to pellet the cells, and resuspend the cells in fresh medium. Count the number of target cells for following experiment and calculate the depletion efficiency.

Acknowledgments

This protocol was adapted from Wang et al. (2009). Ying Zhao is thanked for technical assistance and members of the Sun laboratory are thanked for advice. This work was supported by the NIH.

References

  1. Wang, H. C., Perry, S. S. and Sun, X. H. (2009). Id1 attenuates Notch signaling and impairs T-cell commitment by elevating Deltex1 expression. Mol Cell Biol 29(17): 4640-4652.

简介

BioMag Goat抗大鼠IgG是用多克隆山羊抗大鼠IgG抗体包被的标准BioMag颗粒,并且非常适用于其中将大鼠IgG抗体用作第一抗体的细胞分选方法。 BioMag山羊抗大鼠IgG可用于使用阴性选择从异质细胞群体中分离感兴趣的细胞。 BioMag山羊抗大鼠IgG也可用作酶免疫测定和利用大鼠IgG第一单克隆抗体的放射性测定中的第二抗体。通过阴性选择的富集效率取决于抗原可用性和总细胞群体。 作者有丰富的丰富的小鼠CD4CD8双阴性(DN)胸腺细胞从总胸腺细胞,只含有约2%的DN人口。 通常在一轮富集后,DN人群占总胸腺细胞的80%以上。 然而,骨髓细胞的消耗效率低得多。

材料和试剂

  1. 小鼠总胸腺细胞
  2. BioMag山羊抗大鼠IgG(QIAGEN,50ml,目录号:310104)(QIAGEN,500ml目录号:310107)(1mg/ml)
  3. 大鼠抗小鼠CD4抗体(BD Biosciences/BD Pharmingen TM,目录号:553727)
  4. 大鼠抗小鼠CD8抗体(BD Biosciences/BD Pharmingen TM,目录号:553027)
  5. BioMag暂停
  6. 胸腺细胞
    注意:是否添加抗CD4抗体做耗尽或细胞分选是有争议的,因为一些研究人员声称DN人群的一小部分DN1表达低水平的CD4。

设备

  1. 磁性分离器(BD Biosciences/BD Pharmingen TM,目录号:552311,批次,目录号:0000038241)
    注意:Qiagen还提供适用于不同容器的磁性分离器,如单管(目录号:36910),12管(目录号:36912),15ml/50ml管(目录号:36935)和 烧瓶(目录号:36937)。

程序

  1. 计算
    1. 为了获得最佳结果,使用尽可能稀释的细胞悬液。 通常,以2×10 7总细胞/ml培养基制备总细胞。 由于任何给定的细胞来源将具有独特的纯化要求,因此建议由个体用户确定最佳条件
    2. QIAGEN建议每个细胞使用10-50个磁性颗粒(总细胞群)。 然而,已经证明,每个总胸腺细胞少至2-3个磁性颗粒非常有效地工作。 1mg/ml BioMag悬浮液含有每毫克1×10 8磁性颗粒,相当于每毫升1×10 8磁性颗粒。 因此,所需的BioMag悬浮液的体积是2.0-3.0ml每10 8个总胸腺细胞。

  2. 准备细胞悬液
    1. 将浓度调节为2×10 7总细胞/ml无菌培养基。

  3. 一抗与细胞表面抗原结合
    1. 向细胞中加入适量的一级抗体,并在4℃或冰上孵育30分钟。 为了最大化抗原和抗体之间的结合,倒置管含有细胞和一抗每5分钟几次。 为了获得最佳结果,个体研究者需要优化一级抗体的量。

  4. 清洗BioMag颗粒
    1. 在结合过程中,在适当的无菌培养基或缓冲液中洗涤所需量的BioMag颗粒2-3次。首先,将Biomag颗粒悬浮在含有叠氮化钠的原始缓冲液中,并使用磁分离器(> 20兆欧斯)将磁性颗粒拉到管的侧面。小心地取出原始缓冲液以除去叠氮化钠防腐剂。使用磁选机进行分离2-3分钟。
    2. 重复该程序用低蛋白(5%FCS)缓冲液建议减少非特异性结合 重要:在洗涤步骤中不要离心BioMag悬浮液。离心导致大量聚集和丧失结合活性

  5. 从细胞中去除未结合的一抗
    1. 在200-300×g离心5分钟,并吸上清液。将细胞重悬在无菌培养基中并在200-300×g离心5-10分钟以洗涤细胞。重复洗涤步骤1-2次,每次洗涤使用1ml无菌培养基。

  6. BioMag颗粒结合一抗
    1. 将细胞重悬于适当体积的无菌培养基中,并加入步骤3中制备的洗涤过的BioMag颗粒。无菌培养基和BioMag颗粒的最终体积应保持细胞在2×10 7/ml/ml。 在4℃孵育15分钟。 偶尔旋转反应容器,或在孵育期间将eppendorf管置于旋转的轮上。
    注意:
    1. 为获得最佳结果,总体积应≥1ml(包括BioMag悬浮液和细胞体积)。 当使用体积<1ml时,应加入额外的培养基或缓冲液至最终体积为1ml。
    2. 由于细胞表面随时间变化,磁性颗粒可能从靶细胞脱离,因此不推荐长时间孵育。
    3. 室温(15-25°C)或37°C是某些细胞类型和QIAGEN的最佳选择。
    4. 建议单独确定最佳细胞分选条件。

  7. 将靶细胞与其他不需要的细胞分离
    1. 在4℃下将容器加入磁选机中10分钟。 一旦分离完成,小心地移除上清液而不干扰沉淀到另一个管,上清液含有阴性选择后的靶细胞。
    注意:
    1. 清澈的上清液表明分离完成。
    2. 分离必须在容器垂直放置的情况下进行,以便在烧瓶或管的侧面形成丸粒。 这确保未选择的细胞不会污染磁性颗粒。
    3. 为了获得最佳结果,请重复步骤7一次。 作者强烈建议用至少15分钟的间隔重复步骤7一次,因为任何污染的磁性颗粒将干扰以下实验,特别是细胞分选。

  8. 计数靶细胞
    1. 在200-300×g离心上清液5分钟以沉淀细胞,并将细胞重新悬浮在新鲜培养基中。 计数以下实验的目标细胞的数量,并计算耗尽效率。

致谢

该协议改编自Wang等人(2009)。 感谢Ying Zhao的技术援助,并感谢Sun实验室的成员的建议。 这项工作得到了NIH的支持。

参考文献

  1. Wang,H.C.,Perry,S.S.and Sun,X.H。(2009)。 Id1会削弱Notch信号,并通过提升Deltex1表达来削弱T细胞的承诺。 Mol Cell Biol 29(17):4640-4652。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, H. (2012). Enrichment of Cells of Interest from Heterogeneous Murine Cells with BioMag Goat Anti-rat IgG. Bio-protocol 2(7): e146. DOI: 10.21769/BioProtoc.146.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。