搜索

ROR1 Flow Cytometry
ROR1 流式细胞检测   

评审
匿名评审
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Cancer Cell
Nov 2012

Abstract

ROR1 is a receptor tyrosine kinase family member studied for its roles in development and cancer. Here we describe a protocol for analysis of ROR1 surface expression in acute lymphoblastic leukemia immortalized cell lines by flow cytometry.

Materials and Reagents

  1. Cells (e.g. RCH-ACV cells, Kasumi-2 cells, REH cells, MHH-CALL-2 cells)
  2. FBS
  3. Antibody specific for ROR1 (R&D Systems, catalog number: AF2000 )
  4. Goat IgG (R&D Systems, catalog number: AB-108-C )
  5. Donkey Anti-goat IgG-Phycoerythrin (R&D Systems, catalog number: F0107 )

Equipment

  1. Centrifuge
  2. FACSAria (BD Biosciences)

Procedure

  1. Actively cultured RCH-ACV, Kasumi-2, REH, and MHH-CALL-2 cells were pelleted and washed once in PBS and then resuspended in PBS wash buffer containing 2% FBS (1 million cells in 50 μl of buffer).
  2. 1 x 106 cells were immunostained at room temperature for 30 min with 1 μg of antibody specific for ROR1 or Goat IgG (do not need to rotate the reaction).
  3. Cells were washed 3 times with 500 μl PBS wash buffer.
  4. Cells were stained with Donkey Anti-goat IgG-Phycoerythrin (10 μl Donkey Anti-goat IgG-Phycoerythrin is diluted into 90 μl PBS wash buffer). Incubate at room temperature in the dark for 15 min.
  5. Samples are washed 1x with 500 μl PBS wash buffer and then resuspended in 200 μl PBS wash buffer for analysis.
  6. Samples were analyzed on a BD FACSAria.

Acknowledgments

This protocol was adapted from Bicocca et al. (2012).

References

  1. Bicocca, V. T., Chang, B. H., Masouleh, B. K., Muschen, M., Loriaux, M. M., Druker, B. J. and Tyner, J. W. (2012). Crosstalk between ROR1 and the Pre-B cell receptor promotes survival of t(1;19) acute lymphoblastic leukemia. Cancer Cell 22(5): 656-667.

简介

ROR1是受体酪氨酸激酶家族成员,研究其在发展和癌症中的作用。 在这里我们描述的分析ROR1表面表达的急性淋巴细胞白血病永生化细胞株流式细胞仪的协议。

材料和试剂

  1. 细胞(例如RCH-ACV细胞,Kasumi-2细胞,REH细胞,MHH-CALL-2细胞)
  2. FBS
  3. ROR1特异性抗体(R& D Systems,目录号:AF2000)
  4. 山羊IgG(R& D Systems,目录号:AB-108-C)
  5. 驴抗山羊IgG-藻红蛋白(R& D Systems,目录号:F0107)

设备

  1. 离心机
  2. FACSAria(BD Biosciences)

程序

  1. 将活性培养的RCH-ACV,Kasumi-2,REH和MHH-CALL-2细胞沉淀并在PBS中洗涤一次,然后重悬于含有2%FBS的PBS洗涤缓冲液(50μl缓冲液中1百万个细胞)。 />
  2. 1×10 6个细胞在室温下用1μg特异于ROR1或山羊IgG的抗体免疫染色30分钟(不需要旋转反应)。
  3. 用500μlPBS洗涤缓冲液洗涤细胞3次
  4. 用驴抗山羊IgG-藻红蛋白(10μl驴抗山羊IgG-藻红蛋白稀释到90μlPBS洗涤缓冲液)中染色细胞。 在室温下在黑暗中孵育15分钟
  5. 样品用500μlPBS洗涤缓冲液洗涤1次,然后重悬于200μlPBS洗涤缓冲液中用于分析。
  6. 在BD FACSAria上分析样品

致谢

该方案改编自Bicocca等人(2012)。

参考文献

  1. Bicocca,V.T.,Chang,B.H.,Masouleh,B.K.,Muschen,M.,Loriaux,M.M.,Druker,B.J。和Tyner,J.W。(2012)。 ROR1和Pre-B细胞受体之间的串扰促进t(1; 19)急性淋巴细胞存活 白血病。癌细胞 22(5):656-667。
  • 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: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bicocca, V. T. and Tyner, J. W. (2013). ROR1 Flow Cytometry. Bio-protocol 3(18): e905. DOI: 10.21769/BioProtoc.905.
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的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。