从中央与EAE大鼠中枢神经系统的单核细胞的分离

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Summary

在这个视频中,我们将演示如何从中央与实验性自身免疫性脑脊髓炎大鼠神经系统中分离单个核细胞。

Cite this Article

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Beeton, C., Chandy, K. G. Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE. J. Vis. Exp. (10), e527, doi:10.3791/527 (2007).

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Abstract

无论是研究中枢神经系统(CNS),如实验性自身免疫性脑脊髓炎(EAE,1),或免疫反应的中枢神经系统,如小儿麻痹症,莱姆neuroborreliosis,或神经梅毒感染的一种自身免疫性疾病,它往往是需要隔离的中枢神经系统浸润的免疫细胞。

在这个视频协议,我们将演示如何分离单个核细胞(跨国公司)与EAE大鼠中枢神经系统的。此过程的第一步,需要用生理盐水的解决方案,以确保无血灌溉中枢神经系统血液中的船只仍然的啮齿类动物的心脏灌注。任何血液污染,人为地增加了明显的中枢神经系统浸润跨国公司的数量,可能会改变免疫渗透的明显组成。然后,我们演示了如何删除随后dilaceration大鼠的大脑和脊髓准备一个单细胞悬液。这种悬挂是两层Percoll梯度分离,分离出的跨国公司。洗涤后,这些细胞,然后准备接受任何需要的程序。

使用此过程中分离出的单个核细胞是可行的,可用于电,流式细胞仪(FACS),或生物化学。如果该技术是在无菌条件下进行(在组织培养罩中使用无菌文书)的细胞还可以生长在组织培养液中。一个特定的细胞群,可进一步纯化要么磁选程序或一个流式细胞仪。

Protocol

  1. 深麻醉大鼠。喷雾用70%乙醇,用PBS做10分钟的心脏灌注细胞从血管中删除(切开右心房和左心室灌注通过)。
  2. 取出含有50毫升的冰冷PBS管的脑和脊髓和地点。在70毫米的细胞过滤器放置在一个10厘米的培养皿含有10毫升冰冷的PBS的菜切的大脑和脊髓。记者通过细胞过滤器使用1毫升无菌注射器推杆背面的每一块器官。收集到50毫升管上冰的单细胞悬液。用PBS洗净细胞过滤器和添加到管,直到解决的办法是明确的。
  3. 离心390克为8-10分钟
  4. 重悬细胞20毫升PBS + 30%Percoll和覆盖到10毫升的PBS + 70%Percoll。
  5. 在390克离心机在室温为20分钟。
  6. 卸下管上的脂肪。从接口收集细胞,用PBS洗两次。计数。

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Discussion

此过程中,为所有涉及活体动物的程序,必须由您机构的动物使用及护理委员会批准。我们建议,兽医师或兽医技术员在执行第一次心肌灌注,以确保有足够的麻醉水平是程序之前和期间的动物,和动物不进行不必要的痛苦或困扰。

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Materials

Name Type Company Catalog Number Comments
PBS, 1x, sterile Reagent GIBCO, by Life Technologies 14190-250
Cell strainer, 70 um Tool Fisher Scientific 08-771-2
Percoll Reagent Sigma-Aldrich P1644

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References

  1. Beeton, C., Chandy, K. G. Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis. Journal of Visualized Experiments. 5, Forthcoming.

Comments

13 Comments

  1. hi this is liela from iran i am working on my propozal in pastuor institute in tehran . i and my profesor D.r kadivar isolated mononuclear cell from umbilical cord blood term in hospital end then inject to a 1²0gr rat for homing,and determine or detect them whit PCR technics but we have some difficulty?can u help me?  

    Reply
    Posted by: Anonymous
    July 6, 2008 - 2:39 AM
  2. Hi Liela, I am not sure I can help but will certainly try. What kinds of problems did you meet with your protocol? How did you inject the cells (intravenously, intraperitonealy)? How many cells did you inject? How long after injection did you look for the cells? Where did you look for the cells? Many cells will stay stuck in the very thin capillaries of the lungs so you would have to inject several million (5-10 million) to detect them afterwards. A main concern is the change in species. Did you do anything to prevent the rat's immune system from kiling the human cells you injected? This killing can happen very fast and may be the main reason why you don't detect any cells. Christine

    Reply
    Posted by: Anonymous
    July 10, 2008 - 3:12 PM
  3. Hi Christine, This is an excellent video and your technique is very impressive. My name is Yoyo, a post-doc at Imperial College, London, UK. I have been trying to isolate "sub-cells" (probably MNCs) from Rat left ventricles. The PhD student who left now used to just isolating the LV, followed by mincing with razor blades and then digested in buffer containing collagenase II at a shaking water bath (37C). After 4x15min digestion, cells were filtered through both 70 & 40um cell strainers and spun down. Cell pellets were then lysed with RLT buffer containing B-ME for RNA extraction. However, the quality of cells was usually low (determined by Trypan Blue) and thus the RNA yield was rather inconsistent. When I took over this project, I thought I could use Ficoll gradient to remove dead cells. It seemed to work, but the amount of cells and the quality of RNA were rather low (²60/²80 ratio was 1.4-1.6). I did more research and found that Percoll would be more suitable if isolating cells of species other than human. I then followed a protocol obtained from another post-doc working at a different campus of Imperial College. Her protocol is as follows: re-suspend cell pellets in 1²ml 1.08²g/ml Percoll, and prepare Percoll gradient in a 50ml tube loading from the bottom of a total of 1²ml each of 1.050 and 1.08² (with cells) g/ml Percoll. The tube is then centrifugated at ²000g at room temperature for 30 minutes. This should result with debris/lipids at the top, the fibroblasts and cardiomyocytes at the interface between 1.050 and 1.08², while the pellets will be red blood cells and more debris. As I still strain my digested cells with both 70 % 40um cell strainers and my mincing with blades should have damaged cardiomyocytes to a large extent, I don't think there should be any or many cardiomyocytes left in the interface. I would very much appreciate your expertise and help on this isolation. Is it correct to follow her protocol? I think the Percoll gradient of her and yours are similar to each other, except that you overlay cell suspension in ²0ml 30% Percoll onto 10ml 70% Percoll while her was not really overlaying and it seems to be 1.050g/ml Percoll to be at the bottom instead. I would be most grateful if you could give me some advice on this isolation. I look forward to hearing from you soon. Cheers,   Yoyo

    Reply
    Posted by: Yoyo L.
    March 31, 2009 - 12:54 PM
  4. Dear Dr Beeton, first, thanks for your excellent video.Could i ask you to tell me how can i make 30% and 70% percoll and the percentage of PBS.

    regards

    Reply
    Posted by: Anonymous
    December 18, 2011 - 4:35 AM
  5. I am not sure what your question is. These are simple solutions:
    For the RPMI + 30% Percoll, you need 30% Percoll and 70% RPMI.
    For the PBS + 70% Percoll, you need 70% Percoll and 30% PBS.

    Reply
    Posted by: Anonymous
    December 19, 2011 - 2:31 PM
  6. Dear Dr Beeton, thanks for your reply.

    Reply
    Posted by: Anonymous
    December 23, 2011 - 4:53 AM
  7. Hello Dear Christine Beeton
    Thanks for your useful video. I would be grateful if you response my question: In this video when you work with Mycobacterium tuberculosis H37RA for making it thin powder, you didn't wear mask, is it safe for you?

    Regards

    Reply
    Posted by: Anonymous
    January 21, 2012 - 8:11 AM
  8. Hello Dear Christine Beeton
    I do apologize, above question is about your another video (Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis), but I wrote it in this page, wrongly. I would be grateful if you response me.

    Best Regards

    Reply
    Posted by: Anonymous
    January 21, 2012 - 1:26 PM
  9. Hi Dear Dr Beeton

    Could we use Ficoll gradient instead of Percoll for isolation mononuclear cells from the CNS?

    Reply
    Posted by: Anonymous
    April 10, 2012 - 4:16 AM
  10. I don't see any reason why you couldn't use Ficoll instead of Percoll as long as the gradient densities are maintained.
    Christine

    Reply
    Posted by: Anonymous
    April 13, 2012 - 3:28 PM
  11. I am grateful for your response.

    Reply
    Posted by: Anonymous
    April 13, 2012 - 5:18 PM
  12. Hello Dear Dr Beeton

    I don't access to the your very useful article, so I don't know the speed of centrifuge for two following steps: 1. preparing the pellet of cells in 50ml PBS
    ². make the mononuclear cells layer by using percoll gradient

    I would be grateful if you help me, it is very necessary for my thesis.

    With Best Regards

    Reply
    Posted by: shahram p.
    July 31, 2012 - 3:50 AM
  13. Dear Dr Beeton -

    Thank you for the nice instructions.

    How many cells is it possible to isolate from a naive rat without e.g. EAE?

    Thank you!

    Kind regards
    Dr. Anders Abildgaard
    Denmark

    Reply
    Posted by: Anders A.
    July 30, 2013 - 7:45 AM

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