JoVE Biology

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

1, 1, 1, 2

1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)

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    Summary

    Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

    Date Published: 7/09/2008, Issue 17; doi: 10.3791/773

    Cite this Article

    P. Matheu, M., Sen, D., Cahalan, M. D., Parker, I. Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging. J. Vis. Exp. (17), e773, doi:10.3791/773 (2008).

    Abstract

    Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

    Protocol

    1) Remove both femur bones from one mouse

    1. Use dissection scissors to cut away muscle and expose the femur bone above and below the joints (knee and hip). Grasp the center of the femur with dissection tweezers and cut above and below the joints to leave as much of the epiphysis intact as possible.
    2. Clean off as much muscle as possible using small dissection scissors. Transfer the femur into a dish of RPMI.
    3. All procedures should be carried out in the hood from this point on, using only sterile media, instruments, pipette tips and culture dishes.

    2) Sterilize Femur bones:

    1. In the hood, transfer bones from the RPMI to a small culture dish filled with 70% ethanol on ice.
    2. Allow bones to soak in ethanol for 5 minutes on ice.

    3) Collect Bone marrow under sterile conditions:

    1. Transfer bones to a small culture dish containing sterile filtered Primary DC culture media.
    2. With sterile tweezers, hold the bone over a discard dish or tube, and with sterile scissors, cut each epiphesis (ends of the bone) off. The cut should expose bone marrow which is bright red in the center of the bone.
    3. With a sterile syringe (26-28 gauge needle), suck up about 100-200 mL of sterile media.
    4. Hold the bone with the sterile tweezers over a fresh dish of sterile media and insert the needle into one side of the bone. Position the tip of the needle near the top of the bone and slowly wash the bone out with the sterile media. If you are in the center of the bone, the needle should slide in easily.
    5. The bone marrow washes out, either in small pieces or as a single piece. It should be flushed out of the bone and into the dish of sterile media.
    6. Repeat this step as needed to completely wash the marrow out of the bone.
    7. When the bone is clean, it will be white and translucent.
    8. Repeat this procedure with the other femur bone. Discard each femur bone once it is clean.

    4) Re-suspend and Spin down Bone Marrow cells:

    1. Transfer cells to a sterile falcon tube.
    2. If marrow is still intact, very gently pipette media up and down in the dish, to break up the marrow into a single cell suspension. This may take several minutes and should be done slowly to avoid killing cells by sheer force.
    3. Centrifuge cells as you would any other mammalian cells.

    5) Cell Lysis:

    1. Remove the tube from the centrifuge after the spin is done and pour media off (decant) into a small waste (50 mL falcon tube) in the hood.
    2. Resuspend pellet by gently flicking the bottom of the tube.
    3. Using sterile filtered water and 10x DPBS or 10x PBS, carry out a water lysis to remove the red blood cells (RBCs) using the following volumes:
      • 900 mL sterile filtered water
      • 100 mL 10x DPBS or 10x PBS
    4. Add 100 mL of 10x PBS 5-10 seconds after adding the water. It is very important do this immediately so that only the RBCs are lysed. It is a good idea to have both pipettes filled and ready.
    5. Add 5 mL – 10 mL of sterile DC basic media.

    6) Count Cells:

    1. In the hood, mix the 6 to 11 mL of bone marrow cells by gently swirling the tube with a slight inversion, a 45 degree tilt (not so that the media touches the top of the tube)
    2. Remove 10 mL of media into a sterile tube for counting.
    3. Re-cap the cells and centrifuge again.

    7) Plating Dendritic cells:

    1. Cells should be plated at a density of 1 x 106/mL.
    2. Remove cells from centrifuge, decant media and re-suspend cells in the appropriate amount of Primary DC Media for plating.
    3. Place in tissue culture incubator. 

    8) Culture Care and Maturation:

    Always check cultures before adding more media or using in any experiments. Check the general health of cells and look for potential contamination.

    DAY 0: Dendritic cells plated.

    DAY 3: Remove 75% of the media and non-adherant cells and add back Primary DC media.

    DAY 6: After Initial Cell Plating: Re-plate the cells using the following procedure:

    1. Remove media, which should contain some non-adherant DCs at this point, and place in a sterile 50 mL falcon tube, leaving plates very slightly wet (you don’t want the adherent cells to dry out)
    2. Add 3 mM EDTA in PBS to each plate and allow to sit for 5 minutes.
    3. Hold the plate at a 45 degree angle and gently pipette media up and down against the bottom of the plate to gently dislodge non-adherant cells.
    4. EDTA/PBS should become thicker indicating cells are being collected from the bottom of the plate.
    5. After several minutes of this, cell mixture can be transferred to the 50 mL falcon tube.
    6. Spin all cells after performing this procedure for each plate.
    7. Count and plate the cells at a density of 1 x 10^6 per plate in Secondary DC Media in new sterile 10 cm culture dishes. Return cells to tissue culture incubator.

    DAY 10-11

    Mature DCs ready for stimulation/antigen loading.

    9) Dendritic Cell Stimulation:

    1. Use LPS (lipopolyscaccharide) in combination with desired peptide for activation and peptide loading. Depending on your source of LPS, different stimulation conditions may give better results. Additionally, the desired amount of peptide for DC presentation should be optimized. Stimulation conditions in this protocol were 100 ng/mL LPS and 100 ug/mL OVA (ovalbumin). 
    2. Load antigen/treat with LPS for 18-24 hours prior to harvest/use.

    Dendritic Cell Media: Sterile filter after everything has been added

    Please see the discussion section for a review of different dendritic cell culture conditions and for the resulting dendritic cell phenotype. These culture conditions are designed to produce mature dendritic cells that rapidly home to draining lymph nodes, 18-24 hours after subcutaneous injection and present antigen efficiently.

    Primary DC Media: Sterile filter after everything has been added

    • 500 mL IMDM (remove 55-60 mL for 500mL final volume)
    • 50 mL heat inactivated FBS
    • 5 mL of 200 mM L-Gln, final concentration of 2 mM
    • 100 IU/mL Penicillin and 100 mg/mL streptomycin (5 mL of 10,000 IU/mL Pen and 10,000 mg/mL Strep stock)
    • 50 uM B-Me (14.3 M B-Me stock, dilute 1:100 in chemical hood, use 35 ul per 100 mL media for 50 micromolar concentration)
    • 20-30 ng/mL GM-CSF
    • 100-400 IU/mL IL-4 roughly 10- 40 ng/mL

    SecondaryDC media for maturation*

    100 ng/mL TNF-alpha (add to Primary DC media)

    * Note: GM-CSF + IL-4 should produce immature dendritic cells. Using GM-CSF + IL4 (400 IU/ul) + TNF-alpha (100 ug/mL) will create cells that resemble mature monocyte derived dendritic cells. Mature dendritic cells are known not to take up antigen as efficiently as immature dendritic cells and cultures may have endogenous TNF-a released from stromal cells4.

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    Discussion

    Dendritic cells are key mediators of the adaptive immune response and the most efficient antigen presenting cell characterized to date. Methods for human and mouse dendritic cell culture in the literature differ in the type of cytokines used to influence the development of different dendritic cell types. Notably, GM-CSF, flt3L, IL4, IL13, TNF-alpha and IFN-gamma are used in different combinations to produce mature, immature, inflammatory and steady-state like murine bone marrow derived dendritic cells in vitro3-7. Here, we present a simple method for the production of mature murine dendritic cells that are capable of homing to draining lymph nodes after subcutaneous injection, presenting antigen and activating na ve T cells. The resulting dendritic cell population is typically >85% CD11chigh with inducible expression of CD80/86 upon LPS activation. The addition of TNF-alpha to the culture on day 7 is optional and produces a more mature phenotype8. The maintenance of in vitro Langerhans dendritic cell cultures, TNF-alpha is an important survival factor but does not stimulate the cells to mature9. It should be noted that TNF-alpha from endogenous sources (likely stromal cells) has been reported to be present in the culture media7. Other successful methods for imaging subcutaneously injected dendritic cells include positive selection for CD11c+ cells from the spleen as well as concurrent labeling and activation of the endogenous dendritic cell population1, 2, 10. Production of CD11chigh dendritic cells from bone marrow is a robust method that consistently produces large numbers of dendritic cells capable of antigen presentation in vivo, important in imaging the process of adaptive immune system activation11, 12.

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    Disclosures

    Acknowledgements

    National Institutes of Health Kirchstein Fellowship predoctoral fellowship AI-64128 (MPM), GM-41514 (MDC), GM-48071 (I.P.)

    References

    1. Miller, M. J., Safrina, O., Parker, I., Cahalan, M. D. Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes. J Exp Med. 200, 847-856 (2004).
    2. Henrickson, S. E., et al. T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation. Nat Immunol. 9, 282-291 (2008).
    3. Jefford, M., et al. Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli. Blood. 102, 1753-1763 (2003).
    4. Inaba, K., et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 176, 1693-1702 (1992).
    5. Lutz, M. B., et al. Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF. J Immunol. 169, 3574-3580 (2002).
    6. Xu, Y., Zhan, Y., Lew, A. M., Naik, S. H., Kershaw, M. H. Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking. J Immunol. 179, 7577-7584 (2007).
    7. Inaba, K., Swiggard, W. J., Steinman, R. M., Romani, N., Schuler, G. Isolation of dendritic cells. Curr Protoc Immunol. 3, (2001).
    8. Winzler, C., et al. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J Exp Med. 185, 317-328 (1997).
    9. Koch, F., et al. Tumor necrosis factor alpha maintains the viability of murine epidermal Langerhans cells in culture, but in contrast to granulocyte/macrophage colony-stimulating factor, without inducing their functional maturation. J Exp Med. 171, 159-171 (1990).
    10. 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).
    11. Wei, S. H., et al. Ca2+ signals in CD4+ T cells during early contacts with antigen-bearing dendritic cells in lymph node. J Immunol. 179, 1586-1594 (2007).
    12. Castellino, F., et al. Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction. Nature. 440, 890-895 (2006).

    Comments

    23 Comments

    what are the aproximate yeilds of the process, ie total per femur and the number (from one femur) at D6 replating?
    Reply

    Posted by: AnonymousAugust 12, 2009, 3:57 PM

    I usually have around 1² M DCs per femur on day 6, passing the cells using EDTA rather than trypsin (which is generally less efficient, but has other advantages). Typically the yield is higher if I use a younger animal.
    Reply

    Posted by: AnonymousMay 12, 2010, 6:20 PM

    I read in other protocol that 10 ng/ml of GM-CSF (with out IL4) is enough to derive immature dendritic cells from bone marrow. Do we really need to supplement the media with IL-4?
    Reply

    Posted by: AnonymousJanuary 14, 2010, 2:42 PM

    IL-4 is not necessary, but the resulting DCs will generally be different. Here is a publication that details this:

    " Influence of interleukin-4 on the phenotype and function of bone marrow-derived murine dendritic cells generated under serum-free conditions."

    Wells JW, Darling D, Farzaneh F, Galea-Lauri J.
    Reply

    Posted by: AnonymousMay 12, 2010, 6:14 PM

    Have you tried splitting the cells when they're still immature? I am worried that splitting them would activate them and/or kill a large number of cells...
    Reply

    Posted by: AnonymousMay 12, 2010, 3:36 PM

    Hi Joanna,

    I typically do split the cells while they would be considered immature. This likely leads to a low level activation, but I have not defined the exact nature of the activation status after splitting the cells. In my experience large numbers of cells do not die off after splitting. Prolonged exposure to trypsin or a calcium chelator could result in die off of cells.
    Reply

    Posted by: AnonymousMay 12, 2010, 6:11 PM

    Great, do they usually re-attach normally to the plate? I didnt use trypsin, but i scraped them for splitting, and they are floating around today... im not sure if they're still alive or not!
    Thanks!!
    Reply

    Posted by: AnonymousMay 13, 2010, 10:02 AM

    Hi, yes they should be reattached to the plate by now if they were healthy.
    Reply

    Posted by: AnonymousMay 13, 2010, 12:36 PM

    The video is great, but can you please remove the absolutely tedious music from it? It is so destracting and makes it very difficult to concentrate.
    Reply

    Posted by: AnonymousSeptember 17, 2010, 6:12 AM

    Hello:

    I am slightly confused. Your video says to use 300 mM EDTA, but your protocol below says to use 3mM EDTA. Is this a typo?
    Reply

    Posted by: AnonymousApril 1, 2011, 2:26 PM

    Can you please provide the supplier/manufacturer of the Cytokines (IL-4, GM-CSF, TNFa)?
    thanks
    Reply

    Posted by: AnonymousJune 16, 2011, 9:54 AM

    R&D Systems
    Reply

    Posted by: AnonymousJune 16, 2011, 11:00 AM

    What is the difference between using the EDTA to remove the DCs and using a cell scraper? A ²008 JOVE protocol "Culture of myeloid DCs from bone marrow precursors" uses a cell scraper. Just wondering if there is a reason for using EDTA over a scraper?
    Reply

    Posted by: Greg P.June 5, 2012, 10:54 AM

    Sorry for the late reply. You could use either method, i.e. either EDTA or scraper. Depending on your experiment and the needed number of cells, gentle pipeting up and down 5-10 times using a 5 ml pipet can also give you a good number of DCs. Instead of a scraper you can use a 5 ml syringe and 18G needle to draw in and then flush out media to the bottom of the plates to dislodge DCs. DCs are very sensitive to change. So another tip is to keep the DC plates towards the back of the incubator, so that opening/closing of doors dŒs not change the ambient temperature too much. Hope this helps. - D. Sen
    Reply

    Posted by: Debasish S.June 25, 2012, 8:34 PM

    Could you please sent me an copy of this article which help me out in my PhD. I am culturing with ²0ng/ml of GM-CSF. Is that ok for growing the dendritic cells isolated from murine bome marrow.
    Reply

    Posted by: srinath g.June 22, 2012, 8:13 AM

    ²0 ng/ml is the standard that we use for bone marrow preparation. If you want more mature DCs you can use more upto say 30 ng/ml.
    Reply

    Posted by: Debasish S.June 25, 2012, 8:28 PM

    Hi, I was wondering if you could share or explain what your FSC SSC plot/gate look like? I am not sure what population to gate on the plot.
    Reply

    Posted by: kristin l.November 19, 2012, 3:28 PM

    Hello, for plating the precursor cells, the video mentioned if I am correct 5x10 6/ml and the written protocol is "Cells should be plated at a density of 1 x 106/mL." which one would you recommend.
    About the EDTA I am confused after reading a previous reply. DŒs EDTA reduce the number of DC? So, if I need a large number of cells, you would recommend pipeting up and down or the 5 ml syringe and 18G needle?
    Thanks
    Reply

    Posted by: Luis d.April 18, 2013, 5:53 PM

    Hi Luis. I recommend plating at 1M/mL for the first plating of the cells. If you plan to collect them within a day or two (activation) you can plate them at a higher density. EDTA may be pipetted against the plate (held at about a 45' angle) during the harvesting stage. EDTA helps remove DCs that are adherent to the plate, this will increase your number of collected DCs. Be sure to be gentile at this stage and use a large pipette, not a small gauge needle for collection of cells. EDTA or scrapping is not necessary for collecting DCs and may lower your purity if your culture has a high percentage of fibroblasts. The needle is used to flush the bone marrow - you may use an 18 guauge or smaller as I did in the video. Insulin syringes are handy and most people have them around so I demonstrated the technique with this gauge (²6 1/²).
    Reply

    Posted by: Melanie M.April 19, 2013, 1:48 AM

    Thank you very much for your quick reply!
    Reply

    Posted by: Luis d.April 20, 2013, 10:34 AM

    Hi, I was wondering which mice strain have you used in this experiment?
    Reply

    Posted by: anjali c.April 5, 2015, 11:26 PM

    Hi Anjali,

    In this example we used wild-type C57BL/6J animals. This protocol can be used for most wt animal strains. If you have a transgenic animal with a specific defect you may need to modify growth conditions for optimal results.

    Good luck!
    Reply

    Posted by: Melanie M.April 6, 2015, 1:02 PM

    Hi. Can you tell me why you chose this strain for the experiment?
    Reply

    Posted by: Nymisha N.April 6, 2015, 10:54 PM

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