EAE Ratlarda Merkez Sinir Sistemi Mononükleer Hücreleri İzolasyon

Biology

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Summary

Bu video sıçanlarda deneysel otoimmün ensefalomyelit ile merkezi sinir sistemi mononükleer hücreleri izole etmek için nasıl göstermektedir.

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

Deneysel otoimmun ensefalomiyelit (EAE, 1), ya da bir enfeksiyon, çocuk felci, Lyme neuroborreliosis, ya da nörosifiliz olarak MSS, bağışıklık yanıtı gibi merkezi sinir sistemi (MSS) yönelik bir otoimmün hastalık eğitim olsun, genellikle MSS-infiltre bağışıklık hücrelerini izole etmek için gerekli.

Bu video protokol EAE ile sıçan CNS (ÇUŞ) mononükleer hücreleri izole etmek için nasıl göstermektedir. Bu prosedürün ilk adımı MSS sulama kan damarlarının kan kalmasını sağlamak için tuzlu su çözeltisi ile kemirgen kardiyak perfüzyon gerektirir. Herhangi bir kan kontaminasyon yapay görünür MSS-infiltre ÇUŞ sayısı artacak ve bağışıklık sızmak görünür kompozisyonu değiştirebilir. Daha sonra, tek bir hücre süspansiyonu hazırlamak için sonraki dilaceration sıçan beyin ve omurilik nasıl çıkarılacağını göstermektedir. Bu süspansiyon ÇUŞ izole etmek için bir iki katmanlı Percoll degrade ayrılır. Yıkadıktan sonra, bu hücreler daha sonra gerekli tüm prosedür geçmesi hazırız.

Bu yordamı kullanarak izole edilen mononükleer hücreler canlı ve elektrofizyoloji, flow sitometri (FACS), ya da biyokimya için kullanılabilir. Tekniği steril koşullar altında yapılır (bir doku kültürü kaputu steril alet kullanarak) hücreleri de doku kültürü ortamında yetiştirilen olabilir. Belli bir hücre popülasyonu daha fazla manyetik ayırma işlemleri ya da bir FACS kullanılarak temizlenmiş olabilir.

Protocol

  1. Derinden sıçanlarda uyutmak. % 70 etanol ile Sprey ve kan damarları hücreleri (sol ventrikül ile sağ kulakçıklar ve serpmek kesim) kaldırmak için 10 dakika süreyle PBS ile kardiyak perfüzyon.
  2. Buz gibi soğuk PBS içeren 50 ml'lik bir tüp içinde beyin ve omurilik ve yer çıkarın. Buz gibi soğuk PBS 10 ml içeren bir 10 cm petri yerleştirilen 70 mm hücre süzgeç beyin ve omurilik kesin. 1 ml steril bir şırınga pistonu geri kullanarak hücre süzgecinden her organın parçası basın. 50 ml tüp içine buz üzerinde bir tek hücre süspansiyonu toplayın. Hücre süzgeç PBS ile yıkayın ve çözüm netleşene kadar tüp eklemek.
  3. G. 390 8-10 dakika santrifüj
  4. 10 ml PBS + 70% Percoll üzerine 20 ml PBS +% 30 Percoll ve bindirme hücrelerin tekrar
  5. 390 g oda sıcaklığında 20 dakika santrifüjleyin.
  6. Tüpün üstüne yağ çıkarın. Arayüzü hücreleri toplamak ve PBS ile iki kez yıkayın. Sayın.

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Discussion

Bu prosedür, canlı hayvanlar ile ilgili tüm prosedürler, kurumun hayvan kullanımı ve bakımı komitesi tarafından onaylanmış olması gerekir. Biz bir veteriner hekim veya bir veteriner teknisyeni, hayvan öncesi ve işlem sırasında verilen anestezi yeterli bir düzeyde sağlamak için ilk kalp perfüzyonları yaparken mevcut olması ve hayvanların gereksiz yere acı veya sıkıntı tabi olmadığını tavsiye ederiz.

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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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