प्रतिरक्षा कोशिकाओं के भीतर murine aortas Cytometry विश्लेषण फ्लो
Summary
इस कागज प्रवाह cytometry आधारित aortas की प्रतिरक्षा संरचना की जांच विधि प्रस्तुत करता है. कागज भी एक अतिरिक्त तकनीक है कि आसपास adventitia और पोत दीवार अलग से जांच करने की अनुमति देता है दिखाता है. इस विधि संभावनाओं को खोलता है महाधमनी leukocytes के phenotypical विश्लेषण के प्रदर्शन और atherosclerosis के अध्ययन के लिए कई प्रतिरक्षाविज्ञानी assays लागू.
Abstract
Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a “mock” channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.
Protocol
Discussion
यहाँ, हम murine aortas प्रतिरक्षा सेल संरचना की जांच के लिए एक प्रवाह cytometry आधारित विधि प्रस्तुत करते हैं. इस विधि का प्रमुख लाभ के लिए एक एकल कोशिका के स्तर पर महाधमनी प्रतिरक्षा कोशिकाओं का विश्लेषण और महाधमनी l…
Disclosures
The authors have nothing to disclose.
Acknowledgements
HL55798 PO1 (के.एल. करने के लिए) और अमेरिकन हार्ट एसोसिएशन वैज्ञानिक विकास अनुदान 0525532U (उदा): इस काम के स्वास्थ्य के राष्ट्रीय संस्थानों अनुदान द्वारा समर्थित किया गया.
Materials
| Material | Catalog Number | Company |
| Collagenase XI | C7657 | Sigma-Aldrich |
| Hyaluronidase | H3506 | Sigma-Aldrich |
| DNase I, type 2 | D4527 | Sigma-Aldrich |
| Collagenase I | C0130 | Sigma-Aldrich |
| Collagenase II | LS004174 | Worthington Biochemical Corporation |
| Elastase | LS002292 | Worthington Biochemical Corporation |
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