और चूहे में मैक्रोफेज की कमी पुनर्गठन
Summary
मैक्रोफेज homeostasis में एक केंद्रीय भूमिका निभाते हैं और कई ऊतकों में विकृति. यहाँ प्रस्तुत प्रोटोकॉल घट मैक्रोफेज के लिए तरीकों का वर्णन<em> Vivo में</em, अस्थि मज्जा रेस्पायरेट्रस से polarized मैक्रोफेज पाने, और adoptively चूहों में मैक्रोफेज स्थानांतरित. इन तकनीकों में भूमिका का दृढ़ संकल्प है कि polarized मैक्रोफेज स्वास्थ्य और रोग में खेलने की अनुमति देते हैं.
Abstract
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 IFNγ 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).
Protocol
Discussion
मैक्रोफेज phagocytic कोशिकाओं है कि प्रतिरक्षा प्रणाली में एक महत्वपूर्ण भूमिका निभाते हैं. वे सहज प्रतिरक्षा प्रतिक्रिया शुरू करने और अर्जित प्रतिरक्षा प्रतिक्रिया निर्देशन के लिए जिम्मेदार हैं. आमतौर ?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
यह काम एक 'रिसर्च सहायता में अनुदान' है Crohn है और कनाडा के बृहदांत्रशोथ फाउंडेशन से LMS, जो / गैस्ट्रोएंटरोलॉजी कैनेडियन स्वास्थ्य / अनुसंधान Crohn है 'एस के एसोसिएशन और कनाडा नई अन्वेषक पुरस्कार के बृहदांत्रशोथ फाउंडेशन के एक कनाडा के एसोसिएशन द्वारा समर्थित है के द्वारा समर्थित किया गया था.
Materials
| Name of the reagent | Company | Catalogue number |
| Clodronate-containing liposomes | Clodronateliposomes.org | |
| PBS-containing liposomes | Clodronateliposomes.org | |
| Sterile PBS pH7.4 | Invitrogen | 14190 |
| IMDM | Invitrogen | 12440 |
| Fetal Calf Serum (FCS) | Invitrogen | 12483 |
| acetic acid | BDH Aristar | BDH3092-500MLP |
| Monothioglycerol (MTG) | Sigma | 96-275 |
| Recombinant murine MCSF | Stemcell Technologies | 02951 |
| Recombinant murine GM-CSF | Stemcell Technologies | 02935 |
| Recombinant murine IL-3 | Stemcell Technologies | 02903 |
| Recombinant murine IFNγ | Stemcell Technologies | 02746 |
| Recombinant murine IL-4 | Stemcell Technologies | 02714 |
| Cell Dissociation Buffer | Invitrogen | 13150-016 |
| Rat anti-F4/80 Antibody | AbD Serotec | MCA497GA |
| Mouse anti-ArgI Antibody | BD Transduction Laboratories | 610708 |
Table 1. Specific reagents used in this protocol.
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