이 작품은 박사 짐 후진타오에 CIHR과 CF 캐나다 보조금 의해 CF 캐나다로 라훌 Kushwah에게 수여 박사 학생이기 의해 재정 지원되었다.

1. Ovalbumin으로 마우스의 Sensitization <ol><li> OVA (학년 V, 시그마, MO)의 솔루션을 준비 한 MG / ML의 농도에서 멸균 PBS에서 (솔루션은 -80 ° C에 저장할 수 있습니다.) </li><li> OVA-명반 혼합물을 준비하기 위해 튜브에서 명반을 가지고 1시 1분의 비율로 튜브를 vortexing하면서 dropwise 패션 OVA 솔루션을 추가합니다. 30 분간 혼합 저어과 믹싱 후 바로 사용하십시오. </li><li> 1 ML의 주사기를 사용하여, 마우스 복막 캐비티로 혼합물의 0.2 ML 주사 2 주 후에 다시 주사를 반복합니다. </li></ol> 2. FITC 라벨 Ovalbumin와 마우스의 비강 도전 <ol><li> OVA-명반의 두 번째 주사 후 칠일은 마우스 OVA-FITC와 intranasally 도전을받을 준비가 된 것입니다. </li><li> -80에서 1 밀리그램 / ML 및 aliquots의 저장소의 농도에서 멸균 PBS를 사용 OVA-FITC (시그마)의 솔루션 ° C를 준비 </li><li> 50 ML 팔콘 튜브의 하단과 화학 H의 멸균 거즈를 놓으십시오ood는 거즈에 Aerrane의 5 ML에 추가합니다. 생쥐를 마취하기 위해, 약 5-10초위한 튜브로 동물을 직접. </li><li> 직립 자세 및 멸균 피펫 팁, 드롭 현명한 패션의 생쥐의 nares 진입 OVA-FITC 솔루션의 피펫 100 μl를 사용하여 anesthetized 동물을 잡아. </li><li> 앞으로 이틀 동안 OVA-FITC와 비강 도전을 반복합니다. </li></ol> 3. 단일 세포 폐로부터 정학 및 배수 림프절의 작성 <ol><li> Euthanyl의 intraperitoneal 주사를 이용하여 생쥐를 희생. </li><li> 직류 분석을 복잡 수있는 대식 세포 오염을 줄이기 위해, 폐 세척이 수행됩니다. 폐를 세척하려면 마우스 tracheas는 잠시 카테터와 주사기로 노출되어 cannulated하고, 5 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산)과 함께 얼음처럼 차가운 PBS를 사용하여 더 낮은 호흡 기관을이 폐포 공간에서 세포를 제거하는 3 번 씻어서있다. </li><li> 10 U / ML hepar를 포함하는 PBS의 10 ML과 폐가 Perfuse심장의 우심실 통해에서 폐 vasculature에서 혈액 세포를 제거합니다. 폐 혈액 세포의 대부분의 제거는 지표로, 흰색으로 바뀝니다까지 perfusions을 수행합니다. 이것은 말초 혈액의 오염 세포를 제거하려면 특히 중요합니다. </li><li> 폐를 해부하고 mediastinal 림프절 (MLN)를 제거하고 별도의 접시에있는 림프절을 넣으십시오. (FITC 형광 반응의 담금질을 방지하기 위해 나가기 위해서 조직의 노출을 최소화하기 위해보십시오.) </li><li> 배양 접시에 폐를 놓고 가위를 사용하여 말하다. 다음 37 25 분 동안 폐가 소화 ° collagenase 활동을 중지하려면 다음 5 분 동안 250 U / ML Collagenase D (로슈)와 추가 EDTA (에틸렌 다이아 민 테트라 초산) (10 MM 최종 농도)를 사용하여 C #. </li><li> 100 μm의 셀 스트레이너를 통해 소화 폐의 조각을 통과하고 적혈구를 제거하는 hypotonic 용해를 수행합니다. 단일 세포 현탁액은 DC가의 자세한 분석을위한 준비가되었습니다. </li><li> MLN, T에서 단일 세포 현탁액을 준비하기 위해서는셀 스트레이너를 통해 긴장이어서 위에서 설명한대로 collagenase와 미세 바늘 및 다이제스트를 사용하여 MLN를 완화. </li></ol> 4. DC위한 염색법은 성숙 / 마이 그 레이션을 평가하기 위해 마커 <ol><li> 폐의 DC가를 파악하기 위해, 염색법는 CD11c 및 CD11b에 대해 수행됩니다. 또한 성숙을위한 평가, 염색법은 DC가가 성숙을 받다으로 upregulated되는 CD86과 CD80를 위해 수행됩니다. MLN에서 CD11c + CD11b + OVA-FITC + 세포는 허파로부터 OVA기도 도전에 대응 MLN로 마이 그 레이션 폐 DC가로 식별됩니다. </li><li> 10 × 10 6 세포 / ML FACS 버퍼에 (1 % FBS 및 1mM EDTA (에틸렌 다이아 민 테트라 초산)과 PBS)와 FACS의 염색법에 대한 세포 현탁액 / 관 나누어지는 150 μl의 농도에서 세포를 일시 중지합니다. 폐 DC가의 확인 내용은 다음과 같은 얼룩이 필요합니다 : 흠없는 제어 (OVA-FITC에 노출되지 생쥐의 폐 세포), OVA-FITC 제어, CD11c 단일 제어, CD11b 단일 제어, CD11b+ CD11c + 이중 제어, CD11b + OVA-FITC + 이중 제어, CD11c + OVA-FITC + 이중 제어, MHC II OVA-FITC + CD11c + CD11b + MHCII에 대해 묻다 단일 제어, CD86 단일 제어, CD80 단일 제어 및 샘플 + , OVA-FITC + CD11c + CD11b + CD86 + 및 OVA-FITC + CD11c + CD11b + CD80 +. </li><li> , DC 마이 그 레이션을 평가 MLN에서 단일 세포 현탁액의 절대 셀 계산을 수행하고 이후에 다음과 같은 튜브를 준비하려면 : CD11c 흠없는 제어 (OVA-FITC에 노출되지 생쥐에서 MLN 세포), OVA-FITC 제어, + 단일 제어, CD11b를 + 이중 제어, CD11b + OVA-FITC + 이중 제어, CD11c + OVA-FITC + 이중 제어 및 CD11c + CD11b + OVA-FITC + 샘플. </li></ol> 5. 대표 결과 기도 알레르기 염증을 유발해서 intraperitoneal sensitization에 필요한 시간은 지점이 중요하며 그림 1에 묘사된대로 실시해야합니다. 기도 알레르기 염증의 유도를 확인하기 위해 intraperitoneal sensitization과기도 OVA의 도전을 이어, 일부 쥐들이 희생 수 있으며 그림 2와 같이 histological 분석은 폐 섹션에서 수행 할 수 있습니다. 염증성 세포의 존재는 Hematoxylin &amp; Eosin 얼룩으로 확인하실 수 있습니다 (그림 2A) 및 점액 생산의 존재는 정기 - 산 - 쉬프 얼룩 (그림 2B)로 평가됩니다. 다 합해서 이것이 OVA-sensitized 생쥐 8 OVA 도전에 따라 알레르기기도 염증의 유도를 확인합니다. 반대로, 염분 취급 생쥐의 폐 부분은 어떤 점액 생산의 부재와 함께 염증 무료 것으로 예상된다. 또한 다음 OVA 도전, 폐 DC가 unde<html> rgo의 성숙과 배수 림프절에 대한 후속 마이 그 레이션. sensitized 및 OVA 맡고 생쥐에서 mediastinal 림프절 (MLN)에서 CD11c + 세포의 분석은 식염수 - 취급 생쥐 (그림 3A)에 비해 CD11c + 세포의 높은 비율을 보여주 것으로 예상된다. 또한, CD11c + CD11b의 절대 셀 카운트의 분석 OVA-sensitized과 도전 생쥐의 MLN에서 + OVA-FITC + 세포 (몇 배 더 높은 IE) 상당히 높은 수를 보여주 것으로 예상되는 염분 처리된 생쥐 (의 대응 그림 3B). <img alt="그림 1" src="/files/ftp_upload/4014/4014fig1.jpg" /> 그림 1. ovalbumin의 유도 (OVA) FITC 라벨이 ovalbumin (OVA-FITC)와기도 도전과 함께 생쥐에서 유도된 알레르기기도 염증에 대한 실험적 프로토콜. fig2.jpg &quot;/&gt; OVA기도 도전 다음에 그림 2. OVA sensitization은 ()에 표시 폐 섹션 Hematoxylin &amp; Eosin 염색법에 의해 식별과 같은기도 알레르기 염증의 유도에 이르게하고 또한 폐의 정기 산성 쉬프의 염색법으로 식별기도의 점액 생산에 이르게 (B)와 같이 섹션. <img alt="그림 3" src="/files/ftp_upload/4014/4014fig3.jpg" /> 그림 3. OVA-FITC 도전은 허파에서 mediastinal 림프절 (MLN)에 직류 마이 그 레이션을 유도합니다. () 유동세포계측법의 플롯은 컨트롤이나 OVA-sensitized 생쥐의 MLN에서 CD11c + 세포의 비율을 묘사. (B) 염분이나 OVA-sensitized 생쥐의 MLN에서 CD11c + CD11b + OVA-FITC + 세포의 절대 건의. * P &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/4014/4014fig3large.jpg" target="_blank">큰 그림을 보려면 여기를 누르십시오</a> .

<ol>
	<li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dendritic+cells+in+the+induction+of+regulatory+T+cells.">Role of dendritic cells in the induction of regulatory T cells.</a> Cell Biosci. 20, (2011).</li><li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+of+dendritic+cell+subsets+and+their+function+in+the+host+immune+system.">Complexity of dendritic cell subsets and their function in the host immune system.</a> Immunology. 133, 409-419 (2011).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+our+breath+away:+dendritic+cells+in+the+pathogenesis+of+asthma.">Taking our breath away: dendritic cells in the pathogenesis of asthma.</a> Nat Rev Immunol. 3, 994-1003 (2003).</li><li>Hammad, H., Lambrecht, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+epithelial+cells:+linking+innate+and+adaptive+immunity+in+asthma.">Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma.</a> Nat. Rev. Immunol. 8, 193-204 (2008).</li><li>Lloyd, C. M., Murdoch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerizing+allergic+responses+in+the+lung.">Tolerizing allergic responses in the lung.</a> Mucosal. Immunol. 3, 334-344 (2010).</li><li>Herz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+murine+animal+models+to+study+the+development+of+allergic+bronchial+asthma.">The relevance of murine animal models to study the development of allergic bronchial asthma.</a> Immunol. Cell Biol. 74, 209-217 (1038).</li><li>Herz, U., Lumpp, U., Daser, A., Gelfand, E. W., Renz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+animal+models+to+study+the+central+role+of+T+cells+in+immediate-type+hypersensitivity+responses.">Murine animal models to study the central role of T cells in immediate-type hypersensitivity responses.</a> Adv. Exp. Med. Biol. 409, 25-32 (1996).</li><li>Nakae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+can+contribute+to+multiple+features+of+ovalbumin-induced+allergic+inflammation+of+the+airways+in+mice.">TNF can contribute to multiple features of ovalbumin-induced allergic inflammation of the airways in mice.</a> J. Allergy Clin. Immunol. 119, 680-686 (2007).</li><li>Guth, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+environment+determines+unique+phenotype+of+alveolar+macrophages.">Lung environment determines unique phenotype of alveolar macrophages.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 296, L936-L946 (2009).</li><li>Kushwah, R., Cao, H., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pulmonary+T+cell+response+to+helper-dependent+adenoviral+vectors+following+intranasal+delivery.">Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery.</a> J. Immunol. 180, 4098-4108 (2008).</li><li>Kushwah, R., Oliver, J. R., Zhang, J., Siminovitch, K. A., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+dendritic+cells+induce+tolerance+in+mice+through+suppression+of+dendritic+cell+maturation+and+induction+of+antigen-specific+regulatory+T+cells.">Apoptotic dendritic cells induce tolerance in mice through suppression of dendritic cell maturation and induction of antigen-specific regulatory T cells.</a> J. Immunol. 183, 7104-7118 (2009).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

여기에 제시된 방법은기도 도전 OVA-FITC의 제공을 바탕으로 폐 DC가를 분석하기위한 유동세포계측법 기반 접근 방식을 제공합니다. 이것은 OVA-FITC을 가지고 감시 따라서 직류 인구가 효과적으로 OVA 유발 알레르기기도 염증 과정 중에기도 면역 반응에 참여하는 사람들입니다 폐동맥 DC가의 선택적 모니터링을 허용합니다. 컨트롤 마우스에서 알레르기기도 염증의 부재에서, MLN에서 확인된 철새 DC가?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Ovalbumin </td><td> 시그마 - 올드 리치 </td><td> A5503 </td></tr><tr><td> 명반 </td><td> 써모 과학 </td><td> 77,161 </td></tr><tr><td> OVA-FITC </td><td> 시그마 - 올드 리치 </td><td> A9771 </td></tr><tr><td> Collagenase D </td><td> 로슈 </td><td> 11088858001 </td></tr><tr><td> 항체 </td><td> E-Bioscience </td><td></td></tr></tbody></table>

analysis of pulmonary dendritic cell maturation and migration during allergic airway inflammation

돌기 세포 (DC가)는 항원에 특정한 T 세포를 활성화하여 적응 면역 반응의 개시에 관여 핵심 선수입니다. DC가가 안정된 상태에서 말초 조직에 존재 있지만 항원 자극에 대한 응답으로, DC가이 항원을 가지고 급속하게 그들이 항원에 대한 T 세포 1,2 응답을 시작 배수 림프절로 마이 그 레이션. 또한 DC가 또한자가 면역 질환뿐만 아니라 알레르기 면역 반응 3 시작에서 핵심 역할을한다. DC가이 폐 환경 4 설정에서 관용의 면역 반응과 유도의 두 개시에 필수적인 역할을한다. 폐 환경은 환경 항원 5의 광대한 배열과 노출로 인하여, 크게 tolerogenic입니다. 그러나 어떤 사람은 알레르기와 천식의 유도로 연결 오차의 휴식,있다. 본 연구에서 우리는기도의 DC 성숙과 미그을 모니터링하는 데 사용할 수있는 전략을 설명sensitization에 사용되는 항원에 대한 반응으로 배급. 기도의 DC 성숙 및 마이 그 레이션의 측정은기도 알레르기 염증시 면역 반응의 동력학의 평가를 허용하며 기본 메커니즘과 함께 이후의 면역 반응의 정도를 이해하는데 도움이됩니다. 우리의 전략 sensitizing 대리인으로 ovalbumin의 사용을 기반으로합니다. Ovalbumin 유도된 알레르 기성 천식은기도 eosinophilia, 폐의 염증과 천식 6,7 중 발견된 상승된 IGE 수준을 재현하기 위해 널리 사용되는 모델입니다. sensitization 후, 마우스는 이해의 ovalbumin기도 DC가 특정 라벨링을 허용 FITC 분류 ovalbumin의 비강 전달에 의해 도전을하고 있습니다. 다음으로 여러 DC 특정 마커를 사용하여, 우리는 이러한 DC가의 성숙을 평가할 수 있으며 유동세포계측법를 채용하여 배수 림프절 자신의 마이 그 레이션을 평가할 수 있습니다.

Watch this Scientific Journal Video about Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation at JoVE.com

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

우리는 ovalbumin 유도된 알레르기기도 염증의 설정에서 ovalbumin에 대한 응답으로 폐 돌기 세포의 성숙 및 마이 그 레이션을 모니터링하는 전략을 설명합니다. 이 전략은 감염의 설정에서 폐 돌기 세포의 이주를 평가하기 위해 수정할 수 있습니다.

알레르기기도 염증 동안 폐 돌기 세포 성숙과 마이 그 레이션 분석

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

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JoVE Journal

Immunology and Infection

14.9K 조회수.  McMaster University, Hamilton. 우리는 ovalbumin 유도된 알레르기기도 염증의 설정에서 ovalbumin에 대한 응답으로 폐 돌기 세포의 성숙 및 마이 그 레이션을 모니터링하는 전략을 설명합니다. 이 전략은 감염의 설정에서 폐 돌기 세포의 이주를 평가하기 위해 수정할 수 있습니다.

비디오: Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

마이 그 레이션의 인간 T 림프구 분리, 문화 및 분석 체외에서</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

연구

면역학 및 감염병학

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

세포 성숙없이 돌기 세포의 효율적인 Transfection을 위해 최적화된 프로토콜

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

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.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Murine Aortas 이내에 면역 세포의 흐름 Cytometry 분석

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

사용 모니터링 수지상 세포 이동<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H 자기 공명 영상

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

생체 내 이미징 방법에 급성 및 만성 염증을 구별하는

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

의 내부 기관 관리<em&gt; 헤모필루스 인플루엔자</em&gt; 마우스 모델에서기도 염증을 연구하는

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

와 감염시 배수 림프절에 쥐 피부 수지상 세포의 마이그레이션을 연구하는 CFSE 기반 분석<em&gt; 소 결핵균</em&gt; Bacille 칼 메트 - GUERIN

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

정밀 절단 라이브 폐 수지상 세포를 시각화하기 위해 마우스 폐 슬라이스

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

분석 Murine 가장자리 지역 B 세포 생체 외에서의 전단 흐름 유도 마이그레이션

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...