Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Immunology and Infection

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Summary

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

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Pino, P. A., Cardona, A. E. Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients. J. Vis. Exp. (48), e2348, doi:10.3791/2348 (2011).

Abstract

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

Protocol

Preparation of reagents

Prepare stock isotonic percoll (SIP), 4 ml per brain, by mixing 9 parts of percoll with one part of 10X HBSS without Ca++ and Mg++.

Prepare SIP at 70% in 1X HBSS without Ca++ and Mg++, 2 ml per brain dispensed into a 15 ml polypropylene conical tube.

Note: It is recommended that percoll should be used at room temperature, if used cold, cells tend to clump and cell separation is less efficient.

Tissue collection and homogenization

Anesthetize mice and perfuse through the left ventricle with ice-cold 1X HBSS. Dissect brain and spinal cord and maintain in RPMI without phenol red until all mice are sacrificed.

Place tissues in 7 or 15 ml dounce homogenizer containing 3 ml of RPMI, gently make a cell suspension with a loose-fitting pestle (A size) followed by a tigh-fitting pestle (B size) to further dissociate the tissues. Complete the volume of the cell suspensions to 7 ml with RPMI.

Gradient set up

Add 3 ml of SIP to the cell suspension to make a final 30% SIP

Slowly layer the 10 mL cell suspension on top of the 70% SIP. This is the most critical step of the procedure, use a pipette-aid set in the gravity mode avoiding mixing of the 70% and 30% solutions. A very clear flat line should be visible at the 70%-30% junction.

Centrifuge 30 min at 500G 18°C. Make sure centrifuge will stop with minimal or no brake so that the interphase is not disturbed.

Using a transfer pipet, gently remove the layer of debris from the top of the tube and collect 2.0-3.0 ml of the 70%-30% interphase into a clean conical tube containing 8 ml of 1X HBSS. Ensure that the percoll containing the interphase is diluted about three fold, mix a few times by inversion and centrifugre 7 min at 500G at 18°C.

Resuspend pellet in 1 ml of cell staining buffer and transfer it to a 1.5 ml tube and wash one more time using a micro-centrifuge at 10,000G 1 min at 4°C.

Antibody staining

Resuspend pellets in 50 μl of purified antimouse CD16/CD32 diluted 1:200 in cell staining buffer to block the Fc binding sites. Incubate over ice for 10 min. Count cells using a hemocytometer.

Add 50 μl of antibody cocktail mix and incubate on ice for 30 min. Each antibody must be first titrated to assess optimal dilution. Use appropriate fluorochrome combinations chosen according the capabilities of laser and filter settings of your particular flow cytometer.

Wash cells in cell staining buffer, resuspend pellets in 100-200 μl of cell staining buffer and analyze immediately in the cytometer, or alternatively cells can be resuspended in 2% paraformaldehyde (PFA) prepared in PBS and stored over-night at 4°C.

Representative Results:

After spinning the gradients, the 70%-30% interphase should have a defined white halo, and quantitation of the cells recovered from a naíve mouse should yield 3-5 x105 cells per mouse (brain plus spinal cord). Inflamed brain might have ranging numbers of inflammatory cells depending of the CNS insult or disease model (Figure 1).

Figure 1
Figure 1. Isolation of mononuclear cells, from naíve and EAE-brain at peak disease. Brain cells suspensions were separated over discontinuous 70%/30% percoll gradients. Cells stained were incubated with Fc-block followed by anti-CD45 antibodies for 30 min on ice. Cells were rinsed in cell staining buffer, and fixed in 2% PFA prior acquisition in an LSR-II. CD45 intensity is measured in the Y-axis, where three distinct populations are visualized. Further characterization of the immune phenotype of CD45hi infiltrating cells is implemented using additional antibodies and subsequently analyzed by flow cytometry.

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Discussion

Analyses of cell surface markers in CNS leukocytes from normal and inflamed mouse tissues has been utilized for several decades1-3. Isolation protocols are based in the separation of microglia and leukocytes by density centrifugation4. Here we describe a fast and effective method of isolating CNS leukocytes using discontinuous percoll gradients. After isolation of cells, staining with various antibodies such as -but not limited to- CD45, CD4, CD8, CD11b, CD19, etc. allows the identification of immune populations that infiltrate the CNS upon induction of a particular pathology. Staining with anti-CD45 antibodies reveals three distinct populations by flow cytometry; (1) CD45 negative population consisting of neuronal cells, astrocytes and oligodendrocytes among others nerve cells, (2) CD45lo or intermediate which contains mostly surveillant microglial cells, (3) CD45hi population consisting of infiltrating hematogenous leukocytes. Studying the contribution of myeloid cells during CNS pathologies has been challenging. However, the combination of various cells surface markers5, 6 and the usage of bone marrow chimeric mice have shown insights into the functional distinctions of myeloid cells during CNS inflammation2, 7, 8. Upon immunization with myelin peptides, an autoimmune reaction is induced and a massive influx of blood cells is recruited to the brain. After this point, there is a wide repertoire of antibodies available to study the phenotype of these cells either by the presence of various cell surface molecules, as well as expression of intracellular proteins such as cytokines or transcription factors.

When homogenizing the tissues, use extreme caution handling the homogenizer with ease. Mononuclear phagocytes are highly susceptible to autolysis. If experiencing clumping of the cells, practice a gentler homogenization protocol and add DNase to the buffer during the homogenization step. Therefore, assess percentage of cell dead in your isolated population. This protocol can be easily modified to add various digesting enzymes, such as collagenase, dispase among others. The yield of cells is usually higher. However some cell surface markers are susceptible to this treatment and therefore will limit their detection by flow cytometry due to their cleavage by proteases. In our protocol, we routinely avoid the usage of digesting enzymes, dissect tissues as quick as possible without compromising the perfusion, and avoid leaving the tissues for long periods of time on ice (> 1hr) prior homogenization. The protocol described has the potential to be applied to gene expression, protein and cell culture of the isolated population.

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Disclosures

No conflicts of interest declared.

Acknowledgements

This work was supported by the National Multiple Sclerosis Society (TA 3021-A1/T to AEC) and the University of Texas at San Antonio.

Materials

Tissue homogenization and Gradients

  • Percoll (Amersham)
  • 10X Hanks’ Balanced Salt Solution, without Calcium and Magnesium (Gibco)
  • RPMI Medium 1640, without phenol red (Gibco)
  • 7-ml or 15-ml Dounce Tissue Grinders (VWR)

Flow cytometry

  • Cell Staining Buffer (Biolegend)
  • Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block), BD Pharmingen
  • Hemacytometer (Fisher)
  • Anti-mouse CD45 (clone 30-F11), CD4 (clone RM4-5), CD19 (clone 6D5), BioLegend
  • 10X Phosphate Buffered Saline, without Calcium and Magnesium (Thermo Scientific)
  • Paraformaldehyde (Sigma Aldrich)
  • Flow cytometry analysis performed with a LSR II (BD Biosciences)

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References

  1. Sedgwick, J. D. Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system. Proceedings of the National Academy of Sciences of the United States of America. 88, 7438-7442 (1991).
  2. Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J. Neurosci. Res. 81, 374-389 (2005).
  3. Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J Immunol. 154, 4309-4321 (1995).
  4. Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat. Protoc. 1, 1947-1951 (2006).
  5. Juedes, A. E., Ruddle, N. H. Resident and infiltrating central nervous system APCs regulate the emergence and resolution of experimental autoimmune encephalomyelitis. J. Immunol. 166, 5168-5175 (2001).
  6. Prinz, M., Priller, J. Tickets to the brain: Role of CCR2 and CX(3)CR1 in myeloid cell entry in the CNS. J Neuroimmunol. (2010).
  7. Mildner, A. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat. Neurosci. 10, 1544-1553 (2007).
  8. Djukic, M. Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology following meningitis in mice. Brain. 129, 2394-2403 (2006).

Comments

19 Comments

  1. hi
    I am phd student in immunology. I want to isolate mononuclear cell from brain and spinal cord but I donot know how many cells I can get from each mouse.
    How many cells did you get per mouse in this protocol?

    Reply
    Posted by: Anonymous
    July 29, 2011 - 2:13 PM
  2. hi
    I am phd student in immunology. I want to isolate mononuclear cell from brain and spinal cord but I donot know how many cells I can get from each mouse.
    How many cells did you get per mouse in this protocol?
    How many cells can we get from healthy mouse?

    Reply
    Posted by: Anonymous
    July 29, 2011 - 2:15 PM
  3. Good afternoon
    Applying the protocol, we get 3-5 x 105 (300,000 ²11; 500,000) cells from a na&#²39;ve mouse (brain plus spinal cord).
    Thanks
    Paula Pino

    Reply
    Posted by: Astrid C.
    July 29, 2011 - 5:09 PM
  4. hi
    How many cells did you isolate from unhealthy mouse?
    if you used collagenase and DNAse wasnot better. I think with collagenase and DNAse you could get more cells.
    with this little cells how did you used this cells for flowcytometry ? how many cells did you usde for flowcytometry?

    Reply
    Posted by: Anonymous
    July 30, 2011 - 3:41 AM
  5. Hello
    - The purpose of this protocol is a rapid isolation of mononuclear cells (like microglia) in 3-4 hours, letting you processing more tissues at a time, but without additional steps (like collagenase and DNAse) because these cells can be easily destroyed.
    - These number of cells can be acquired by flow cytometry....the PDF document of our publication shows a summary of the protocol with corresponding results demonstrating how these method complemented with flow cytometry can distinguish cells from the brain (microglia) from cells from the periphery (leukocytes).
    If you do not have full access to the publication, I can give you a copy of the PDF document.
    Thanks
    Paula Pino

    Reply
    Posted by: Astrid C.
    July 30, 2011 - 11:35 AM
  6. Hello

    Could you please send me a copy of the PDF document with the lymphocyte isolation protocol from brain? My email address is: ernesto.marcos@cigb.edu.cu

    Thanks in advance

    Best regards
    Ernesto

    Reply
    Posted by: Anonymous
    January 11, 2012 - 5:18 PM
  7. Hi Paula, I was wondering if you can send me a copy of the PDF document with the protocol. My email is awchi03@gmail.com. Thank you.

    Anthony

    Reply
    Posted by: Anonymous
    October 4, 2011 - 8:29 AM
  8. Hi Paula,
    I'm a PhD student in Thailand. I plan to isolate macrophages from CNS. Do you mind if I ask you for the PDF document of the protocol? I plan to do the experiment (just try) around the end of January.
    My email address is w_wipawee@yahoo.com
    Thank you and happy new year ²01²

    Wipawee

    Reply
    Posted by: Wipawee W.
    December 29, 2011 - 2:04 PM
  9. Hi Paula,
    two additional questions : what is the approximate weight of your tissue sample (one whole mouse brain + additional spinal cord) , just wondering because the cell yield seems quite high compared to our experience of isolation protocols with and without enzymatic digestion.
    The other question is regarding the viability of the harvested cells - I assume you did Trypanblue-staining and counting - so what is the viability (%) ?
    If you could send me a pdf-copy of your article as well I'd be extremely happy.

    Thanks a lot in advance.
    Karoline
    (I'd be

    Reply
    Posted by: Anonymous
    January 23, 2012 - 3:33 AM
  10. Hello
    I am sorry for the delay to respond your question.
    That is exactly the advantage of our method, cell yield is high compared to other protocols because they imply a lot of steps that make you lose your cells.
    Yes, we do stain cells with Trypan blue, and the viability is 80-90%.
    Please, give me any email address to send you a copy of the pdf document.

    Thanks
    Paula Pino

    Reply
    Posted by: Astrid C.
    January 26, 2012 - 7:30 PM
  11. karoline.mŒller@izi.fraunhofer.de

    Thank you!!

    Reply
    Posted by: Anonymous
    January 27, 2012 - 5:01 AM
  12. Hi Paula,

    If you could send a copy to matt (at) hancocklab (dot) com I would be very appreciative :). Thanks!

    Reply
    Posted by: Anonymous
    February 1, 2012 - 10:46 AM
  13. Hi Paula,
    Could you send me a copy of PDF document of the protocol, too ? ^^
    My email address is seungchang@gmail.com
    Thanks

    Reply
    Posted by: Anonymous
    February 6, 2012 - 11:32 PM
  14. Hi Paula

    Could you, please e-mail me the protocol as well ? My e-mail address is: kklimatcheva@vaccinex.com
    Thank you !

    Katya

    Reply
    Posted by: Anonymous
    February 23, 2012 - 1:36 PM
  15. Hi Dear Dr Cardona
    Could we use Ficoll gradient instead of Percol for isolation of brain and spinal cord mononuclear cells?

    Reply
    Posted by: Anonymous
    April 10, 2012 - 4:08 AM
  16. Hi

    I am not sure if Dr. Cardona had answered your question.
    I will answer it anyways:
    The protocol has been very well standardized using Percoll only.
    In my experience performing other procedures, Ficoll can contain certain levels of endotoxin that can contaminate your samples and interfere with your results.

    I am very sorry for the delay to respond your question.
    Thank you very much

    Paula Pino

    Reply
    Posted by: Astrid C.
    June 30, 2012 - 10:13 PM
  17. Hello Dear Dr Pino
    I am very grateful for your response, it would be very useful for me. I am so sorry for my delay in response your kindness.
    Thank your team

    Reply
    Posted by: Anonymous
    July 18, 2012 - 9:04 AM
  18. I am phd student working on decidual macrophages.I have been trying to isolate Dm's using percoll after enzyme digestion.I am losing the cells and the viability.Can you give me tips to isolate them and may i have your protocol sent to mspriyaa11@gmail.com.

    Thank you

    Reply
    Posted by: shanmuga priyaa m.
    August 25, 2012 - 10:20 AM
  19. Hello,

    I request you to kindly email me a PDF version of this article ? Thanks.

    Manju.

    Reply
    Posted by: Manjunatha V.
    April 17, 2013 - 3:10 AM

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