Name: depletion and reconstitution of macrophages in mice
Uploaded: 2012-08-01T13:00:00
Description: Watch this Scientific Journal Video about Depletion and Reconstitution of Macrophages in Mice at JoVE.com

This work was supported by a 'Grant in Aid of Research' from the Crohn's and Colitis Foundation of Canada to LMS, who is supported by a Canadian Association of Gastroenterology/Canadian Association of Health Research/Crohn's and Colitis Foundation of Canada New Investigator Award.

1. Depleting Macrophages Using Clodronate-containing Liposomes
<ol>
 <li>Liposomes are stored at 4 &deg;C. Two hours prior to injection, remove clodronate-containing liposomes, PBS-containing liposomes, or sterile PBS (injection control) from the refrigerator to allow them to acclimate to room temperature (18 &deg;C).</li>
 <li>Invert the tubes containing liposomes 8-10 times to ensure an even distribution before loading 200 &mu;l into a 1 ml syringe. Attach a 26 gauge needle to the top of the syringe.</li>
 ...

<ol>
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Macrophages are phagocytic cells that play an important role in the immune system. They are responsible for initiating the innate immune response and directing the acquired immune response. Typically, classically activated macrophages are activated by IFN&gamma; or LPS and are responsible for eliminating pathogens and mounting an inflammatory response1. Conversely, alternatively activated macrophages are activated by IL-4 or IL-13 and play a role in debris scavenging and tissue remodeling during the...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Clodronate-containing liposomes</td>
 <td><a href="http://Clodronateliposomes.org" target="_blank">Clodronateliposomes.org</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS-containing liposomes</td>
 <td><a href="http://Clodronateliposomes.org" target="_blank">Clodronateliposomes.org</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile PBS pH7.4 </td>
 <td>Invitrogen</td>
 <td>14190</td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Invitrogen</td>
 <td>12440</td>
 </tr>
 <tr>
 <td>Fetal Calf Serum (FCS)</td>
 <td>Invitrogen</td>
 <td>12483 </td>
 </tr>
 <tr>
 <td>acetic acid</td>
 <td>BDH Aristar</td>
 <td>BDH3092-500MLP </td>
 </tr>
 <tr>
 <td>Monothioglycerol (MTG)</td>
 <td>Sigma</td>
 <td>96-275</td>
 </tr>
 <tr>
 <td>Recombinant murine MCSF</td>
 <td>Stemcell Technologies</td>
 <td>02951</td>
 </tr>
 <tr>
 <td>Recombinant murine GM-CSF</td>
 <td>Stemcell Technologies</td>
 <td>02935</td>
 </tr>
 <tr>
 <td>Recombinant murine IL-3</td>
 <td>Stemcell Technologies</td>
 <td>02903</td>
 </tr>
 <tr>
 <td>Recombinant murine IFN&gamma; </td>
 <td>Stemcell Technologies</td>
 <td>02746</td>
 </tr>
 <tr>
 <td>Recombinant murine IL-4</td>
 <td>Stemcell Technologies</td>
 <td>02714</td>
 </tr>
 <tr>
 <td>Cell Dissociation Buffer </td>
 <td>Invitrogen</td>
 <td>13150-016</td>
 </tr>
 <tr>
 <td>Rat anti-F4/80 Antibody</td>
 <td>AbD Serotec </td>
 <td>MCA497GA</td>
 </tr>
 <tr>
 <td>Mouse anti-ArgI Antibody</td>
 <td>BD Transduction Laboratories</td>
 <td>610708</td>
 </tr>
 </tbody>
</table>
Table 1. Specific reagents used in this protocol.

depletion and reconstitution of macrophages in mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Watch this Scientific Journal Video about Depletion and Reconstitution of Macrophages in Mice at JoVE.com

Depletion and Reconstitution of Macrophages in Mice

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

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