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 JoVE Biology

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

1, 1

1Department of Physiology and Biophysics, University of California, Irvine (UCI)

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    Summary

    In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

    Date Published: 12/04/2007, Issue 10; doi: 10.3791/527

    Cite this Article

    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).

    Abstract

    Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.

    In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure.

    Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

    Protocol

    1. Deeply anesthetize rats. Spray with 70% ethanol and do a cardiac perfusion with PBS for 10 min to remove cells from blood vessels (cut the right atria and perfuse through the left ventricle).
    2. Remove the brain and spinal cord and place in a 50 ml tube containing ice-cold PBS. Cut the brain and spinal cord in a 70 mm cell strainer placed in a 10 cm petri dish containing 10 ml of ice-cold PBS. Press each piece of organ through the cell strainer using the back of a sterile 1 ml syringe plunger. Collect the single cell suspension into a 50 ml tube on ice. Wash the cell-strainer with PBS and add to the tube until the solution is clear.
    3. Centrifuge for 8-10 min at 390 g.
    4. Resuspend the cells in 20 ml PBS + 30% Percoll and overlay onto 10 ml of PBS + 70% Percoll.
    5. Centrifuge at 390 g for 20 min at room temperature.
    6. Remove the fat on top of the tube. Collect the cells from the interface and wash twice with PBS. Count.

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    Discussion

    This procedure, as all procedures involving live animals, must be approved by your institution's animal use and care committee. We recommend that a veterinarian or a veterinary technician be present when performing the first cardiac perfusions to ensure a sufficient level of anesthesia is given to the animal before and during the procedure, and that the animals do not undergo unnecessary pain or distress.

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    Disclosures

    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

    References

    1. Beeton C. and Chandy K.G. Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis, Journal of Visualized Experiments, 5, http://www.jove.com/index/Details.stp?ID=214, doi: 10.3791/214.

    Comments

    13 Comments

    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: AnonymousJuly 6, 2008, 2:39 AM

    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: AnonymousJuly 10, 2008, 3:12 PM

    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

    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: AnonymousDecember 18, 2011, 4:35 AM

    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: AnonymousDecember 19, 2011, 2:31 PM

    Dear Dr Beeton, thanks for your reply.
    Reply

    Posted by: AnonymousDecember 23, 2011, 4:53 AM

    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: AnonymousJanuary 21, 2012, 8:11 AM

    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: AnonymousJanuary 21, 2012, 1:26 PM

    Hi Dear Dr Beeton

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

    Posted by: AnonymousApril 10, 2012, 4:16 AM

    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: AnonymousApril 13, 2012, 3:28 PM

    I am grateful for your response.
    Reply

    Posted by: AnonymousApril 13, 2012, 5:18 PM

    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

    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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