Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Published 11/01/2007
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Summary

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically ≥ 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Cite this Article

Copy Citation

Matheu, M. P., Cahalan, M. D. Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification. J. Vis. Exp. (9), e409, doi:10.3791/409 (2007).

Abstract

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are ≥ 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Protocol

Please read and follow the protocol included in the Miltenyi kit or online at http://www.miltenyibiotec.com/.

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Discussion

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are ≥ 96% pure CD4+/CD3+. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions.

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We wish to thank Rebecca Paquett for the preparation of MACS buffer.

Materials

Name Type Company Catalog Number Comments
MACS Buffer Reagent None None Prepare as described by Miltenyi.
CD4+ T cell Isolation Kit: Untouched Isolation Reagent Miltenyi Biotec 130-090-860 For use with the Miltenyi system.
Sterile Phosphate-Buffered Saline (PBS) Reagent For use in the preparation of MACS buffer.
Corning 35mm Not TC-Treated Culture Dish Other Corning 430588 Used in the preparation of a single cell suspension.
70 micron Cell Strainer Tool BD Biosciences 352350 Used in the preparation of a single cell suspension.
6 mL Syringe Tool Tyco Healthcare, Covidien 8881516051 Used in the preparation of a single cell suspension.
10x Dulbecco’s Phosphate Buffered Saline (DPBS) Reagent Invitrogen 14200-075 For use in RBC lysis.
Sterile Water Reagent Sigma-Aldrich W4502 For use in RBC lysis.

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References

  1. Miller, M. J., Hejazi, A. S., Wei, S. H., Cahalan, M. D., Parker, I. T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node. Proc Natl Acad Sci U S A. 101, 998-1003 (2004).
  2. Matheu, M. P., Deane, J. A., Parker, I., Fruman, D. A., Cahalan, M. D. Class IA phosphoinositide 3-kinase modulates basal lymphocyte motility in the lymph node. J Immunol. 179, 2261-2269 (2007).

Comments

13 Comments

  1. Comment: Typo in water lysis portion of the protocol. Font for microliters translated as mL instead of uL. Use 900 uL of ddH²0 and 100 uL of 10x PBS.

    Reply
    Posted by: Melanie M.
    August 13, 2008 - 12:54 AM
  2. Thank you Melanie. That was a great presentation as we are trying to remove axillary lymph nodes and isolate T-cells for our experiments. Do you know by any chance what is an approximate number of cells (total vs T-cells (CD4+ and CD8+, etc)) can you isolate from axillary lymph from naive, let's say C57 or balb/c mice? We might need about, in best case scenario, at least 5*10^6. I appreciate any help you might provide us with. Good luck! Sincerely, Vyachesalv Palchevskiy  

    Reply
    Posted by: Anonymous
    September 4, 2008 - 1:04 AM
  3. Hi, glad you found this video to be helpful. If you are only taking the axillary nodes and purifying naive cells I would use 3-4 mice to be sure you obtain the 5x10^6 that you need for CD4+ T cells. I would start with 4 since axillary nodes can vary in size. Typically the CD4 to CD8 ratio in a mouse is ²:1, though this varies between animals. If you need more lymph nodes from this region I suggest also taking the brachial nodes which are just underneath the skin, next to the muscle just below the fore'arm' of the mouse, number 5 on this chart: http://www.geocities.com/virtualbiology/lymph².html I hope this helps and best of luck in all of your research!

    Reply
    Posted by: Melanie M.
    September 4, 2008 - 1:23 AM
  4. Thank you. Do you ever digest lymph nodes or spleens to get dendritic cells? For us, looks like it affects viability and changes our FACS profile. Thanks, Vyacheslav Palchevskiy, Ph.D.

    Reply
    Posted by: Anonymous
    November 3, 2008 - 10:31 PM
  5. Dear Mr. Vyacheslav Palchevskiy, You posted a request regarding the isolation of dendritic cells from murine spleen and/or lymph nodes. Dendritic cells are located within the tissue and in order to release them from the tissue context, it is necessary to perform an enzymatic digest. The enzymatic conditions should always be optimized to be strong enough to release the target cells and at the same time remain as gentle as possible to avoid unnecessary cell death. Still, it cannot be circumvented to have higher amounts of dead cells. In case the numbers rise above 10%, I would recommend removing these cells as can be easily achieved using our Dead Cell Removal Kit. It is also well known that the various enzymes used for digesting different types of tissue result in alterations of certain surface marker expression. Sometimes the structure of the surface marker is altered in such a way that the common antibody clone cannot bind anymore because they do not recognize their epitope any longer. Therefore, it is always important to test if the enzyme in use affects the relevant markers of a given experiment or not. Usually dendritic cells require a digest using collagenase IV (e.g. from Roche), subsequently the dendritic cells can be magnetically separated using CD11c Microbeads or else stainings can be performed using one of the fluorochrome conjugated antibodies specific for CD11c. There is no known influence of this digest on the CD11c molecule.
    I hope I could help you, otherwise do not hesitate to contact me again. Kind regards, Lisa Siewe   Dr. Lisa Siewe
    Produktspezialist
    Technical Support

    Miltenyi Biotec GmbH
    Friedrich-Ebert-Straße 68
    514²9 Bergisch Gladbach
    Germany

    Phone +49 ²²04 8306- 830
    Fax +49 ²²04 8306- 89
    macstec@miltenyibiotec.de

    www.miltenyibiotec.com

    Sitz der Gesellschaft/Principal Office: Bergisch Gladbach
    Registergericht/Commercial Register: Amtsgericht Köln, HRB 46171
    Geschäftsführer/Managing Directors: Stefan Miltenyi, Dr. Boris Stoffel
    USt

    Reply
    Posted by: Anonymous
    November 5, 2008 - 9:20 AM
  6. Hello! Thank you for the response (and the excellent answers that have followed). In our hands we have similar results where collagenase digestion reduces cell viability. We obtain more than enough dendritic cells from our single cell suspensions, therefore we avoid using collagenase. It may be particullarly useful in harvesting DCs from more collagen rich tissues (skin etc.). Best wishes, Melanie

    Reply
    Posted by: Anonymous
    November 5, 2008 - 11:59 AM
  7. Dear forum participants I just wanted to comment on the subject of viability of cells after preparation with or without digestion. When following our general protocol for preparation of lymphoid organs without digestions (see our website), one can expect a viability of 90-95% for spleen. When doing a Collagenase digestion for the preparation of dendritic cells, as it is recommended for most of our murine dendritic cell products (e.g. CD11c MicroBeads), a very similar viability rate can be expected. Meaning, we do not observe a considerable difference in viability between digested and non-digested spleen tissue. The advantage of using Collagenase for dendritic cell isolations is, that one receives a higher yield of these rare target cells. I hope this information was helpful for you. Best regards, Frank Hardung - Technical Support – Miltenyi Biotec GmbH

    Reply
    Posted by: Anonymous
    November 27, 2008 - 4:20 AM
  8. ).  In the spleen we noticed if we digest with collagonase A + Dnase it decreases the flow for CD4 CD8 CD44 for example as compared to an undigested spleen - which digest do you recommend and what is the best way to talk with you or someone in your Co.

    Reply
    Posted by: Anonymous
    November 8, 2008 - 9:46 AM
  9. Reply
    Posted by: Anonymous
    February 19, 2009 - 12:25 PM
  10. Well- you seem to have enough time to make funny videos,...-
    Anyway- if you would use the columns the right way (as described in detail in the datasheet) and if you would use a good cell suspension- you can quickly isolate your desired cells. I never had this problem before- it is fast and always works. Never had something as reliable as MACS beads in the lab!

    Reply
    Posted by: Anonymous
    May 7, 2009 - 8:33 PM
  11. How many T cells do you get out of a lymph node, or out of all the lymph nodes of one mouse?

    Reply
    Posted by: Anonymous
    April 21, 2009 - 1:02 PM
  12. I am sorry, but i desagree because it is not possible you present only a video, I believe it is neccesary to present the complete protocol writen and do not only reference to a different link.

    Reply
    Posted by: Honorio T.
    October 29, 2009 - 7:06 AM
  13. hi:
    why i cannot see the vedio

    Reply
    Posted by: Hawazen B.
    May 14, 2010 - 1:38 AM

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