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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, 18634 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

 

I. 抗体

1.        BD mouse Fc Block (BD Pharmingen 553142)

2.        Rat anti-mouse B220 APC (Southern Biotech 1665-11)

3.        Rat anti-mouse CD138 PE (BD Pharmingen 561070)

注意: 上面的抗体已经被作者验证过了并且可能根据作者需要被替换了.

II. 其他材料:

1.        FITC BrdU Flow Kit (BD Pharmingen 559619) * provided in the kit

2.        BrdU (Sigma B5002)

3.        Dulbecco’s Modified Eagle Medium (DMEM) (1X) (Invitrogen 10313-039)

4.        胎牛血清(Invitrogen 11091-148)                             

 

设备:

 

1.        BD LSR II 流式细胞仪

 

步骤:

 

1.        给小鼠腹腔注射1mg BrdU (溶于200ul of 无菌 PBS.

2.        喂小鼠含有0.8mg/ml BrdU 水连续14. 每天需水量不同,并且要用锡箔纸包好避光保存.

注意: 14 天足以用BrdU标记新和成的浆细胞在这个品系老鼠而且不会对老鼠产生毒害作用.

3.        杀死小鼠取出脾脏.

4.        用表面霜化的一对显微镜载玻片通过温和的粉碎脾脏成碎块得到单细胞悬浮液在5ml DMEM.

5.        转移细胞到50ml圆锥形管,4oC 300xg for 5分钟离心.

6.        去除上清不要汗有其他颗粒.

7.        5ml 1x ammonium chloride lysing solution 重悬细胞并冰上放置5分钟.

8.        加入15ml DMEM到细胞中4oC 300xg 离心5 分钟.

9.        去除上清用20ml DMEM 重悬细胞并计数.

10.    重悬2百万的脾细胞用50ul of 1:200 BD Fc block in FACS buffer 冰上孵育30分钟.

11.    200ul PBS洗细胞并4oC 300xg 离心5分钟

12.    去除上清并用含有1:200 anti-mouse B220 APC and anti-mouse CD138 PE100ul FACS buffer重悬细胞.

13.    在室温下孵育细胞15分钟.

14.    Step 11洗细胞.

15.    去除上清并用100ul of BD cytofix/Cytoperm buffer*重悬细胞.

16.    冰上孵育30分钟固定细胞.

17.    1ml of BD perm/Wash Buffer*洗细胞用,4oC 300xg 离心5分钟.

18.    弃上清用100ul of BD Cytoperm Plus Buffer*重悬细胞.

19.    冰上孵育细胞10分钟.

20.     Step 17洗细胞.

21.    100ul of BD Cytofix/Cytoperm Buffer*重悬细胞.

22.     冰上孵育5分钟固定细胞.

23.     Step 17洗细胞.

24.    弃上清用100ul of diluted DNase* (300ug/ml in PBS)重悬细胞.

25.    37oC孵育细胞一个小时来曝光标记的BrdU.

26.     Step 17洗细胞.

27.    弃上清用含anti-BrdU FITC 50ul of BD Perm/Wash Buffer*重悬细胞.

28.    室温下孵育细胞20分钟.

29.     Step 17洗细胞.

30.    1ml of PBS重悬细胞并用流式细胞仪分析染色的细胞.

注意:细胞可以用2%甲醛重悬并在4oC储存过夜.

31.    流式细胞检测分析

1)       Gate on plasma cells (B220intCD138hi)

 

BrdU1.png

 

2)       Gate on BrdU positive and negative cells.

 

     BrdU2.png

 

配方:

 

1.        Ammonium Chloride 10X Lysing Solution (1L)

96g NH4Cl                     

10g KHCO3                                       

3.7g 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

300ul Fetal bovine serum                                   

10ml PBS

 

参考文献

 

1.        Hoyer B.F., Moser K., Hauser A.E., Peddinghaus A., Voigt C., Eilat D., Radbruch A., Hiepe F., Manz R.A. (2004). Short-lived plasmablasts and long-lived plasma cells contribute to chronic humoral autoimmunity in NZB/W mice. Journal of Experimental Medicine 199(11): 1577-84. 

2.        Liu Z., Bethunaickan R., Huang W., Lodhi U., Solano I., Madaio M.P., Davidson A. (2011). Interferon-alpha accelerates murine systemic lupus erythematosus in a T cell-dependent manner. Arthritis and Rheumatism 63(1): 219-29. 

 

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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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