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BrdU (Bromodeoxyuridine or 5-bromo-2’-deoxyuridine) is a synthetic nucleoside that is incorporated into DNA by proliferating cells. This protocol is to be used to incorporate and detect BrdU in murine plasma cells. The plasma cells described in this protocol are formed spontaneously in autoimmune mice (NZB/W mice). Modifications are most likely needed if users intend to label plasma cells in immunized mice.

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[Bio101] In vivo BrdU Incorporation and Detection in Mouse
[Bio101] 小鼠体内BrdU的标记和检测

免疫学 > 免疫细胞功能 > 综合
作者: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, 6/5/2011, 20460 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.81

[Abstract] BrdU (Bromodeoxyuridine or 5-bromo-2’-deoxyuridine) is a synthetic nucleoside that is incorporated into DNA by proliferating cells. This protocol is to be used to incorporate and detect BrdU in murine plasma cells. The plasma cells described in this protocol are formed spontaneously in autoimmune mice (NZB/W mice). Modifications are most likely needed if users intend to label plasma cells in immunized mice.

Keywords: Autoimmune(自身免疫性), Mouse(鼠标), Plasma cells(浆细胞)

[Abstract] BrdU (溴脱氧尿苷 or 5-溴-2’-脱氧尿苷) 是一种人工合成的核苷酸,能通过增殖细胞插入DNA中. 这个方法是用来在鼠浆细胞中标记和检测BrdU . 在这个方法中的浆细胞是在自身免疫的小鼠(NZB/W mice)中自发产生的.如果想要在免疫过的小鼠中标记 浆细胞,修饰时最可能用到的.

Materials and Reagents

  1. Antibodies
    1. BD mouse Fc Block (BD Biosciences, Pharmingen™, catalog number: 553142 )
    2. Rat anti-mouse B220 APC (Southern Biotech, catalog number: 1665-11 )
    3. Rat anti-mouse CD138 PE (BD Biosciences, Pharmingen™, catalog number: 561070 )
      Note: The above antibodies have been tested by the author and may be substituted with the antibodies desired by users.

  2. Other materials
    1. Mice
    2. FITC BrdU Flow Kit (BD Biosciences, Pharmingen™, catalog number: 559619 )
      Note: *Provided in the kit.
    3. BrdU (Sigma-Aldrich, catalog number: B5002 )
    4. 1x Dulbecco’s modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-039 )
    5. Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 11091-148)
    6. Ammonium chloride 10x lysing solution (see Recipes)
    7. FACS buffer (see Recipes)

Equipment

  1. BD LSR II flow cytometer

Procedure

  1. Give the mice one i.p. injection of 1 mg BrdU in 200 μl of sterile PBS.
  2. Feed the mice water containing 0.8 mg/ml BrdU for 14 days. The water needs to be changed daily and be wrapped in aluminum foil to avoid light.
    Note: 14 days are needed to label all the newly synthesized plasma cells with BrdU in autoimmune mice (NZB/W mice) and would not result in noticeable toxic effects in the mice.
  3. Sacrifice the mice and harvest the spleen.
  4. Create single cell suspension by gently smashing spleen pieces with the frosted surface of a pair of microscope slides in 5 ml of DMEM.
  5. Transfer cells into 50 ml conical tubes and spin down cells at 300 x g for 5 min at 4 °C.
  6. Discard the supernatant with aspiration without disturbing pellet.
  7. Resuspend cells with 5 ml of 1x ammonium chloride lysing solution (see Recipes) and incubate on ice for 5 min.
  8. Add 15 ml DMEM to cells and spin at 300 x g for 5 min at 4 °C.
  9. Discard supernatant and resuspend cells with 20 ml of DMEM and count cells.
  10. Resuspend 2 millions spleen cells in 50 μl of 1:200 BD Fc block in FACS buffer (see Recipes) and incubate for 30 min on ice.
  11. Wash cells with 200 μl of PBS and spin down the cells at 300 x g for 5 min at 4 °C.
  12. Discard supernatant and resuspend cells in 100 μl FACS buffer containing 1:200 anti-mouse B220 APC and anti-mouse CD138 PE.
  13. Incubate cells at room temperature for 15 min.
  14. Wash cells as in step 11.
  15. Discard supernatant and resuspend cells with 100 μl of BD cytofix/Cytoperm buffer*.
  16. Incubate for 30 min on ice for fix cells.
  17. Wash cells with 1 ml of BD perm/Wash Buffer* and spin at 300 x g for 5 min at 4 °C.
  18. Discard supernatant and resuspend cells with 100 μl of BD Cytoperm Plus Buffer*.
  19. Incubate cells for 10 min on ice.
  20. Wash cells as in step 17.
  21. Resuspend cells with 100 μl of BD Cytofix/Cytoperm Buffer*.
  22. Incubate for 5 min on ice to re-fix cells.
  23. Wash cells as in step 17.
  24. Discard supernatant and resuspend cells with 100 μl of diluted DNase* (300 μg/ml in PBS).
  25. Incubate cells for 1 h at 37 °C to expose incorporated BrdU.
  26. Wash cells as in step 17.
  27. Discard supernatant and resuspend cells with 50 μl of BD Perm/Wash Buffer* containing anti-BrdU FITC* (1:50 in buffer).
  28. Incubate cells for 20 min at room temperature.
  29. Wash cells as in step 17.
  30. Resuspend cells in 1 ml of PBS and analyzed stained cells with a flow cytometer (run at a rate no greated than 400 events/second).
    Note: Cells can be resuspended in 2% formaldehyde and stored overnight at 4 °C in dark, prior to analysis by flow cytometry.
  31. Flow cytometric analysis
    1. Gate on plasma cells (B220 int CD138hi)


    2. Gate on BrdU positive and negative cells


Recipes

  1. Ammonium chloride 10x lysing solution (1 L)
    96 g NH4Cl
    10 g KHCO3
    3.7 g Na4EDTA
    Add ddH2O to final volume
    Adjust pH to 7.2-7.4 and autoclave
    Add ddH2O 9:1 to make 1x lysing solution
  2. FACS buffer
    300 μl FBS
    10 ml PBS

Acknowledgments

This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).

References

  1. Hoyer, B. F., Moser, K., Hauser, A. E., Peddinghaus, A., Voigt, C., Eilat, D., Radbruch, A., Hiepe, F. and Manz, R. A. (2004). Short-lived plasmablasts and long-lived plasma cells contribute to chronic humoral autoimmunity in NZB/W mice. J Exp Med 199(11): 1577-1584.
  2. Liu, Z., Bethunaickan, R., Huang, W., Lodhi, U., Solano, I., Madaio, M. P. and Davidson, A. (2011). Interferon-alpha accelerates murine systemic lupus erythematosus in a T cell-dependent manner. Arthritis Rheum 63(1): 219-229.

材料和试剂

  1. 抗体
    1. BD小鼠Fc Block(BD Biosciences,Pharmingen TM,目录号:553142)
    2. 大鼠抗小鼠B220 APC(Southern Biotech,目录号:1665-11)
    3. 大鼠抗小鼠CD138PE(BD Biosciences,Pharmingen TM,目录号:561070)
      注意:上述抗体已经被作者测试,可能被用户需要的抗体替代。

  2. 其他材料
    1. 小鼠
    2. FITC BrdU流动试剂盒(BD Biosciences,Pharmingen TM,目录号:559619) 注意:*提供在工具包中。
    3. BrdU(Sigma-Aldrich,目录号:B5002)
    4. 1×Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Invitrogen TM,目录号:10313-039)
    5. 胎牛血清(FBS)(Life Technologies,Invitrogen TM,目录号:11091-148)
    6. 氯化铵10x裂解液(见配方)
    7. FACS缓冲区(参见配方)

设备

  1. BD LSR II流式细胞仪

程序

  1. 给小鼠一个腹膜内。 注射1mg BrdU在200μl无菌PBS中
  2. 饲养含有0.8mg/ml BrdU的小鼠水14天。 水需要每天更换,并用铝箔包裹,以避免光 注意:在自身免疫小鼠(NZB/W小鼠)中需要14天来用BrdU标记所有新合成的浆细胞,并且不会在小鼠中导致明显的毒性作用。
  3. 牺牲老鼠和收获脾脏。
  4. 通过用5ml DMEM中的一对显微镜载玻片的磨砂表面轻轻地捣碎脾脏来产生单细胞悬浮液。
  5. 将细胞转移到50ml锥形管中,并在4℃下以300×g离心5分钟。
  6. 弃去上清液,不要打扰沉淀。
  7. 用5ml 1×氯化铵裂解液(见Recipes)重悬细胞,并在冰上孵育5分钟。
  8. 向细胞中加入15ml DMEM,并在4℃下以300×g旋转5分钟。
  9. 弃去上清液并用20ml DMEM和计数细胞重悬细胞
  10. 在FACS缓冲液(见Recipes)中的50μl1:200 BD Fc块中重悬2百万个脾细胞并在冰上孵育30分钟。
  11. 用200μlPBS洗涤细胞,并在4℃下以300×g离心5分钟。
  12. 弃去上清液并将细胞重悬于含有1:200抗小鼠B220 APC和抗小鼠CD138 PE的100μlFACS缓冲液中。
  13. 在室温下孵育细胞15分钟
  14. 洗涤细胞,如步骤11
  15. 弃去上清液并用100μlBD cytofix/Cytoperm缓冲液*重悬细胞
  16. 在冰上孵育固定细胞30分钟。
  17. 用1ml BD Perm/Wash Buffer *洗涤细胞,并在4℃下以300xg旋转5分钟。
  18. 弃去上清液并用100μlBD Cytoperm Plus Buffer *重悬细胞
  19. 孵育细胞在冰上10分钟。
  20. 洗涤细胞,如步骤17.
  21. 用100μlBD Cytofix/Cytoperm缓冲液*重悬细胞
  22. 在冰上孵育5分钟以重新修复细胞
  23. 洗涤细胞,如步骤17.
  24. 弃去上清液并用100μl稀释的DNase *(300μg/ml PBS中)重悬细胞。
  25. 在37℃下孵育细胞1小时以暴露掺入的BrdU
  26. 洗涤细胞,如步骤17.
  27. 弃去上清液并用50μl含有抗-BrdU FITC *(缓冲液中1:50)的BD Perm/Wash Buffer *重悬细胞。
  28. 在室温下孵育细胞20分钟
  29. 洗涤细胞,如步骤17.
  30. 重悬细胞在1ml的PBS中,并用流式细胞仪分析染色的细胞(以不超过400次/秒的速率运行)。
    注意:在通过流式细胞术分析之前,可以将细胞重悬于2%甲醛中并在4℃下避光保存过夜。
  31. 流式细胞术分析
    1. 在浆细胞上的门(B220 int CD138hi)


    2. 门在BrdU正电池和负电池上


食谱

  1. 氯化铵10×裂解溶液(1L)
    96g NH 4 Cl
    10克KHCO 3
    3.7g Na 4 EDTA 将ddH 2 O添加到最终量
    将pH调节至7.2-7.4,高压釜
    添加ddH 2 O 9:1以制备1x裂解溶液
  2. FACS缓冲区
    300μlFBS
    10ml PBS

致谢

该方案在Anne Davidson博士在Feinstein Institute for Medical Research,NY,USA的实验室中开发或修改。这项工作得到NY SLE基金会(RB),Rheuminations,NIH AI082037和AR 049938-01,NIH(PO1 AI51392和PO1 AI51392的流式细胞术和蛋白质表达和四聚体核)的赠予的支持。

参考文献

  1. Hoyer,B.F.,Moser,K.,Hauser,A.E.,Peddinghaus,A.,Voigt,C.,Eilat,D.,Radbruch,A.,Hiepe,F.and Manz, 短期存活的浆母细胞和长期存活的浆细胞有助于NZB/W小鼠的慢性体液免疫。 J Exp Med 199(11):1577-1584。
  2. Liu,Z.,Bethunaickan,R.,Huang,W.,Lodhi,U.,Solano,I.,Madaio,M.P.and Davidson,A。(2011)。 干扰素-α以T细胞依赖的方式加速小鼠系统性红斑狼疮。 arthritis Rheum 63(1):219-229。
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How to cite this protocol: Liu, Z. (2011). In vivo BrdU Incorporation and Detection in Mouse. Bio-protocol Bio101: e81. DOI: 10.21769/BioProtoc.81; Full Text



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