Isolatie van mononucleaire cellen uit het centrale zenuwstelsel van ratten met EAE

Biology

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Summary

In deze video laten we zien hoe de mononucleaire cellen van het centrale zenuwstelsel van ratten met experimentele auto-immune encephalomyelitis isoleren.

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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

Of het bestuderen van een auto-immuunziekte gericht aan het centrale zenuwstelsel (CZS), zoals experimentele auto-immune encephalomyelitis (EAE, 1), of de immuunrespons op een infectie van het CZS, zoals poliomyelitis, Lyme neuroborreliose of neurosyfilis, is het vaak noodzakelijk om de CNS-infiltrerende immuuncellen te isoleren.

In deze video-protocol waarin we laten zien hoe mononucleaire cellen (MNC) van het CZS van een rat met EAE te isoleren. De eerste stap van deze procedure is een cardiale perfusie van de knaagdieren met een zoutoplossing om ervoor te zorgen dat er geen bloed blijft in de bloedvaten bevloeit het centraal zenuwstelsel. Elke besmet bloed zorgt voor een kunstmatige toename van het aantal schijnbare CNS-infiltrerende multinationals en kan de schijnbare samenstelling van het immuunsysteem infiltreren wijzigen. Vervolgens hebben we laten zien hoe de hersenen en het ruggenmerg van de rat voor latere dilaceratie te verwijderen om een ​​single-cell suspensie te bereiden. Deze schorsing is gescheiden op een twee-lagen Percoll verloop van de multinationals te isoleren. Na het wassen, deze cellen zijn dan klaar voor elke gewenste behandeling te ondergaan.

Mononucleaire cellen geïsoleerd met behulp van deze procedure zijn levensvatbaar en kunnen gebruikt worden voor de elektrofysiologie, flow cytometrie (FACS), of biochemie. Als de techniek wordt uitgevoerd onder steriele omstandigheden (met behulp van steriele instrumenten in een weefselkweek kap) de cellen kunnen ook worden gekweekt in weefselkweek medium. Een bepaalde cel populatie kan verder worden gezuiverd met behulp van magnetische scheiding procedures of een FACS.

Protocol

  1. Diep verdoven ratten. Spray met 70% ethanol en doe een cardiale perfusie met PBS gedurende 10 min om cellen van de bloedvaten (snijd de rechter boezem en perfuseren door het linker ventrikel) te verwijderen.
  2. Verwijder de hersenen en het ruggenmerg en doe dit in een 50 ml buis met ijskoud PBS. Snijd de hersenen en het ruggenmerg in een 70 mm cel zeef geplaatst in een 10 cm petrischaal met 10 ml ijskoud PBS. Druk op elk stukje orgaan door de cel zeef met behulp van de achterkant van een steriele 1 ml spuit zuiger. Verzamel de enkele celsuspensie in een 50 ml buis op ijs. Was de cel-zeef met PBS en toe te voegen aan de buis totdat de oplossing helder is.
  3. Centrifugeer voor 8-10 min op 390 g.
  4. Resuspendeer de cellen in 20 ml PBS + 30% Percoll en overlay op 10 ml PBS + 70% Percoll.
  5. Centrifugeer bij 390 g gedurende 20 min bij kamertemperatuur.
  6. Verwijder het vet op de top van de buis. Het verzamelen van de cellen van de interface en was tweemaal met PBS. Tellen.

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Discussion

Deze procedure, als alle procedures met betrekking tot levende dieren, moet worden goedgekeurd door dier van uw instelling gebruik en onderhoud commissie. Wij adviseren een dierenarts of een veterinaire technicus aanwezig zijn bij het uitvoeren van de eerste cardiale perfusie naar een voldoende niveau van de anesthesie te waarborgen wordt gegeven aan het dier voor en tijdens de procedure, en dat de dieren ondergaan onnodig pijn of angst.

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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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