저자는 편집 지원 앤 다론 (Daron)과 피에르 루이 Loyher을 감사드립니다, 두 광자 현미경 및 지원을 사육 쥐에 대한 동물 시설 &quot;NAC&quot;와 카밀 Baudesson에 대한 지원은 Plateforme Imagerie Pitié-Salpêtrière (PICPS). 부여 계약에 따라 이러한 결과로 이어지는 연구는 유럽 공동체의 일곱 번째 프레임 워크 프로그램에서 자금 지원을받은 (FP7 / 2007-2013) N 304810 ° - 공습을, n은 대학 학자 피에르 등의 마리 퀴리에서 INSERM에서 241,440 - Endostem을, ° &quot;등장 &quot;라에서&quot; &quot;에서,&quot;리그 contre 르 암 협회는 라 공들인 쉬르 르 암을 부어 &quot;과에서&quot;직원은 국립 드 라 공들인 &quot;프로그램의 출현 2012 (ANR-EMMA-050). PH는 라 &quot;리그 contre 르 암&quot;에 의해 지원되었다.

참고 : 모든 실험 프로토콜 프랑스 동물 실험 윤리위원회의 승인 번호 A-75-2065에 &quot;서비스 보호 등 상테 짐승 같은, ENVIRONNEMENT&quot;에 의해 검증되었다. 샘플 크기는 실험의 재현성을 보장하기 위해 선택, 동물 윤리 규정의 3R에 따라 있습니다. 마우스 1. 준비 <ol><li> 마취 <ol><li> 촬상의 짧은 기간 (1 시간 미만)를 들면, 케타민 (100 ㎎ / kg) 및 크 실라 진 (10 ㎎ / kg)을 함유하는 식염수 용액 200 μL의 복강 내 주사하여 마우스를 마취시키다. </li><li> 또한, 영상의 긴 기간 동안 (4 시간까지), 적응 마스크를 통해 O 2 / N 2 O의 70/30 혼합물에서 기화 이소 플루 란 2.5 %의 흡입으로 마우스를 마취시키다. </li><li> 참고 :이 방법은보다 안정적인 무의식을 제공하고 약물의 시리얼 복강 내 주입을 방지 할 수 있습니다. </lI&gt; <li> 마우스가 마취되면, 니퍼와 발바닥의 자극을 통해 무의식을 확인하십시오. </li></ol></li><li> 혈관 염색 / 추가 염색 <ol><li> 혈관의 얼룩 : 꼬리 정맥에 정맥 내 Rhodamin-200 μL의 덱스 트란 (2 MDA 식염수 완충액 중 10 ㎎ / ㎖)을 주입한다. </li><li> 호중구 염색의 경우 : 정맥 꼬리 정맥에 생리 식염수 100 ㎕에 (클론 1A8) Ly6G-PE의 2 μg의 주입. </li></ol></li><li> 고정화 <ol><li> 고정의 경우, 현미경 및 마취 가스 흡입기에 맞게 맞춤 제작 한 정위 홀더를 사용합니다. 참고 : sterotactic 소유자는 동물의 머리를 체결 할 수있는 기타 이동식 및 적응 두 개의 금속 플레이트 브래킷, 하나는 고정되어와 함께 금속 지원입니다. </li><li> 호흡 운동으로부터 격리를 보장하기 위해 고정 플레이트 사이의 마우스의 머리를 설치합니다. 고정 플레이트와 SK를 스트레칭 머리 사이에 귀를 조인치 안정성과 동물의 호흡 복지를 확인합니다. </li></ol></li><li> 두피를 제거 <ol><li> 조심스럽게 눈과의 접촉을 피하고, 멸균 주걱으로 두피의 젖은 머리 에탄올을 70 %를 사용합니다. </li><li> 완전히 정면 뼈 정수리 뼈의 후방에서 두피를 제거하기 위해 멸균 가위와 집게로 피부를 잘라. 광범위한 출혈을 방지하기 위해 눈과 귀에서 3mm까지 멀리 할 것. 두개골 (골막)을 덮고있는 느슨한 결합 조직을 제거합니다. 따뜻한 PBS 적신 종이 타월로 남아있는 머리카락을 세척 할 것. </li></ol></li><li> 집중 시스템 설정 <ol><li> 직접 분포를 제어하기 위해 작은 게이지 바늘을 이용하여 두개골에 수술 글루 18mm 직경의 고무 링을 붙여. (30 μL가 충분해야한다) 누출을 방지하기 위해 고무 링의 전체 둘레를 붙인다. </li><li> PBS 적신 종이 타월로 링에서 마지막으로 한 번 두개골을 청소 한 다음의 완전한 침수 37 ° C PBS를 추가두개골. 안구 건조를 방지하기 위해 마우스의 눈은 0.9 % 염화나트륨 용액 또는 특정 안연고 방울을 추가한다. 목적에 따라 정위 홀더를 드래그합니다. </li><li> 약 직접 전송 현미경을 통해 안구를 사용하여 필드를 놓습니다. </li></ol></li></ol> 2. 두 광자 이미징 취득 <ol><li> 선택적으로 680에서 1080 nm의 동조, NIR 광의 140fs 펄스를 제공 사파이어 레이저, 레이저 전력 제어를위한 음향 광학 변조기 : 티타늄과 결합 다 광자 현미경을 사용한다. 2 다이크로 익 미러 (565 nm에서 690 nm의), 610분의 565 및 550분의 500 대역 통과 필터의 조합 (3 형광 채널의 동시 녹음 가능) 3 외부 비 descanned 감지기 (NDD)를 포함하는 구성을 사용하고, 20 (NA = 1) 침수 목적 × 계획 아포 크로 매트 485 짧은 패스 필터. </li><li> anaes 좋은 homeothermy 있도록 가열 챔버를 설정thetized 마우스., 참고 : 온도는 더 나은 안정성을 위해 세션을 이미징 전에 1 시간을 설정할 수 있습니다. 우리의 경우, 32 ° C는 마취 마우스의 최적의 체온 (36 ~ 37 ° C)를 얻기 위해 선택되었으며, 4 시간까지 유지된다. 이 온도는 사용하는 (디지털 프로브가이 점을 확인하는 데 사용할 수있는) 시스템에 적응 될 수있다. 또한, 사용 된 가열 챔버는 블랙 / 어둡고 외광 오염을 방지 시스템 (목적 및 전동 플레이트)의 일부를 커버한다. </li><li> 시스템을 켭니다 <ol><li> 사파이어 레이저, 다음 전체 현미경 시스템 : 티를 켭니다. 수집 소프트웨어를 시작합니다. 870 nm의 레이저 파장 범위를 설정. 녹음에 적합한 NDD을 선택합니다. 참고 : 우리의 경우, 두 번째 고조파 발생 (SHG) (19)과 시안 형광 단백질 (ECFP)가 485 짧은 패스 필터 후 NDD에 의해 감지됩니다. 550분의 500 대역 통과 필터 및 rhodamin 덱스 트란이 t에 의해 검출 된 후 ECFP도 NDD 의해 검출그는 NDD 610분의 565 대역 통과 필터 후. </li></ol></li><li> 인수 설정을 조정합니다 <ol><li> 소프트웨어의 &quot;라이브 취득&quot;촬상 명령을 사용하여 현미경 방향 제어를 사용하여, 로다 민계 ECFP 신호 영역에 초점을 조정한다. 최소한의 광 손상 최상의 신호 대 잡음비를 구하는 최소 레이저 파워를 설정한다. 주 : 사용 된 전력 설정은 관심 영역의 깊이에 따라 달라집니다. 일반적 AOM 설정은 15 mW의 주위를 넘어 대물 레이저 파워를 나타내는 약 20 %로 설정된다. 그것은 각각의 NDD이 사진 표백 및 사진 손상을 감소하는 PMT가에 이득 전력을 증가하는 것이 좋습니다. 전형적인 획득 설정 1.58 μS 하나의 화소 지속 시간과 스캔 및 배율 1.5 512 X 512 픽셀의 해상도이다. </li></ol></li><li> 관심의 선택 영역 <ol><li> 기록 파라미터가 설정되면, 다시 &quot;라이브&quot;취득 커맨드를 사용실시간으로 모니터링 할 목적 (X, Y, Z)를 선택하는 필드 조직을 조사. 참고 : 일반적으로, 우리는 혈관 및 실질 영역 모두를 포함하는 필드를 선택했다. </li><li> &quot;위치&quot;소프트웨어 명령을 사용하여 필드를 등록합니다. 참고 :이 명령은 X, Y, 다른 먼 필드의 Z 좌표를 기억합니다. </li><li> 선택과 같은 절차를 사용하여 (3까지) 관심의 추가 필드를 등록합니다. </li><li> 선택 배율 필요한 볼륨에 따른 제 기억 위치에 &quot;Z-스택&quot;소프트웨어 명령으로 3D의 Z-스택을 설정합니다. 제 깊은 위치하고 높은 위치를 외운다. 깊이 레이저 파워 정규화. 주 : 여러 부수적 필드 촬상을 위해, 우리는 12 μm의 두께의 체적을 확보하기 위해 각 필드의 3 μm 인 Z-5 단계로 구분하여 슬라이스를 획득. 이러한 설정은 연구 셀의 타입에 적응 될 수있다. </li></ol></li><li> 시작 획득 <ol><li> 선택220; 시계열 &quot;소프트웨어 명령 및 시간 간격의 지속 기간과 사이클의 수를 선택한다. &quot;실험을 시작&quot;소프트웨어 명령을 선택하여 필요한 시간의 경과와 기간에 따라 시간 경과 영상을 시작합니다. 주 : 우리의 경우는 30 분 실시간을 나타내는 60 사이클 동안 하나의 볼륨 각각 30 초를 획득. 작은 시간 경과는 혈관에서 고속 셀 압연을 추적하기 위해 수행 될 수있다. 이 경우, Z 얇게 스택 및 2 개 이하의 상이한 필드에 동시에 기록 될 수있다. </li><li> 셋업의 정기적 인 모니터링. <ol><li> 항상 의식이 있는지 확인하기 위해 동물을 모니터링 할 수 있습니다. 참고 : 수염 또는 동물의 다리의 동요는 부흥의 지표입니다. 동물의 호흡이 불규칙적 인 경우에, 이소 플루 란의 양이 다소 감소 될 수있다. 인수시 강한 조직 드리프트는 마우스 부흥의 지표입니다. </li><li> 목표를 확인하는 것은 제대로 담근다. 37 ° C PBS의 betwee 갱신N 개의 인수 (30 분). 참고 : 인수시 신호의 프로그레시브 실종 PBS 누설의 지표입니다. </li></ol></li></ol></li><li> 절차의 끝 <ol><li> 필요한 동영상이 기록되면, 신속하게 심폐 소생술 전에 자궁 전위에 의해 동물을 안락사. 주 : 상처 치유는 길이 촬상을 수행 할 수있는 가능성을 제한하는 24 ~ 48 시간에 딱지의 형성을 유도. </li></ol></li></ol> 3. 데이터 분석 <ol><li> 드리프트 보정 <ol><li> 3D 자동 추적 Imaris 비트 평면의 소프트웨어를 사용합니다. </li><li> Imaris에 3D 이미지 시퀀스를 엽니 다. 선택 명령을 &quot;발견&quot;, 균일 분야에서 배포 가능하면, 4 개의 앵커 포인트의 최소 모든 시점 수동 추적 (마우스 클릭 + 이동)을 생성합니다. </li><li> &quot;편집 트랙&quot;탭에서 &quot;올바른 드리프트&quot;명령을 사용하여 드리프트 보정을 적용합니다. </li><li> t에 대한 확인그는 X에있는 보정, Y, 특히 Z에서의 품질은 artefactual 흔들림을 방지 할 수 있습니다. 참고 : 보정이없는 만족스러운 경우, 다른 고정 점을 시도하거나 분석에서 비디오를 제외합니다. </li></ol></li><li> 셀 추적 <ol><li> 명령 &quot;새로운 관광 명소를 추가&quot;를 사용 및 알고리즘 설정 &quot;시간이 지남에 트랙 명소&quot;를 선택합니다. 다음 단계로 이동합니다. </li><li> &quot;소스 채널&quot;명령을 사용하여 관심있는 전체 채널 중 하나를 자동 세포 추적 절차를 적용합니다. 10 μm의 개체를 설정합니다 (즉, 세포) 직경. 다음 단계로 이동합니다. 참고 : 선택은 측정 된 신호의 객체와 특이도의 차이에 따라 달라집니다. 즉, 전체 채널에 자동 추적은 검출 된 신호가 관심 대상에 특정한 경우에만 가능하다. SHG 및 ECFP 모두 485 짧은 패스 필터 후의 NDD 의해 검출되기 때문에,보다 구체적으로는 트래킹 및 오 후 NDD ECFP 의해 기록 신호를 이용하여 효율적인 것00/550 대역 통과 필터. 클러스터 또는 조밀 한 포장 객체는 3.1.2 단계와 유사 개별 개체의 수동 선택이 필요합니다. </li><li> &quot;품질&quot;필터 유형을 선택 불특정 객체의 추적을 제외 할 임계 값을 설정합니다. 다음 단계로 이동합니다. </li><li> 두 지점 사이의 &quot;최대 거리&quot;와 &quot;갭의 크기&quot;의 정확한 매개 변수와 함께 &quot;브라운 알고리즘 모션&quot;을 선택합니다. 트랙 생성을 확인합니다. </li><li> 트랙이 자동으로 계산되면, &quot;트랙 기간&quot;필터 유형을 선택 인 트랙 (4 시점을 나타내는)보다 짧은 120 초를 제거하기 위해 임계 값을 설정합니다. </li><li> 필수 : 자동 추적 한 결과, 트랙의 품질을 확인. <ol><li> 객체가 트랙 경로를 따라 확인하는 시간 줄을 풀다. , &quot;편집&quot;탭에서 사용할 수 트랙 포인트 보정의 옵션을 사용하지 않는 경우 : &quot;삭제&quot;명령 (모든 트랙 페이지를 삭제하여 허위 또는 부적절한 트랙을 삭제OINT 개별적으로) &quot;연결 해제&quot;명령을 사용하여. 트랙 경로가 불연속 인 경우, 누락 된 시점 (마우스 클릭 + 이동)를 추가 주어진 트랙에서 모든 지점을 선택하고 사용하는 &quot;편집 트랙&quot;탭에서 명령을 &quot;연결&quot;. </li></ol></li></ol></li><li> 정량 매개 변수 <ol><li> , &quot;환경 설정&quot;명령에서 &quot;통계&quot;탭을 선택 목록에서 관심있는 동적 매개 변수를 선택합니다. 선택 명령 및 생성 된 엑셀 파일을 저장 &quot;파일을 모든 통계를 내 보냅니다.&quot; 참고 : 여러 동적 매개 변수가 직접적 트랙 길이, 순간 속도, 평균 속도로 소프트웨어에서 얻은, 그리고 직진을 추적하고 있습니다. 운동성 계수 6은, (20)을 결정할 수 있지만, 소프트웨어에 의해 직접 제공되지 않는다. </li><li> 각 셀의 경우, 시간 (ΔT)의 각 기간에 대한 변위의 평균 계산 (30 초 시간 경과 영화를, ΔT는 30, 60, 90 초, 등이 될 것이다).시간 간격의 함수로서 사각형의 평균 변위를 플롯. 곡선의 직선 부분의 기울기를 계산함으로써 운동성 계수를 구하는. </li></ol></li></ol>

<ol>
	<li>Ovchinnikov, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Gal4-dependent+transgenes+in+cells+of+the+mononuclear+phagocyte+system+labeled+with+enhanced+cyan+fluorescent+protein+using+Csf1r-Gal4VP16/UAS-ECFP+double-transgenic+mice.">Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice.</a> J Leukoc Biol. 83, 430-433 (2008).</li><li>Parihar, A., Eubank, T. D., Doseff, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+macrophages+regulate+immunity+through+dynamic+networks+of+survival+and+cell+death.">Monocytes and macrophages regulate immunity through dynamic networks of survival and cell death.</a> J Innate Immun. 2, 204-215 (2010).</li><li>Geissmann, F., Jung, S., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+monocytes+consist+of+two+principal+subsets+with+distinct+migratory+properties.">Blood monocytes consist of two principal subsets with distinct migratory properties.</a> Immunity. 19, 71-82 (2003).</li><li>Robbins, C. S., Swirski, F. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+splenic+reservoir+monocytes+and+their+deployment+to+inflammatory+sites.">Identification of splenic reservoir monocytes and their deployment to inflammatory sites.</a> Science. 325, 612-616 (2009).</li><li>Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+the+immune+system:+progress,+pitfalls+and+promise.">Dynamic imaging of the immune system: progress, pitfalls and promise.</a> Nat Rev Immunol. 6, 497-507 (2006).</li><li>Charo, I. F., Ransohoff, R. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+bone+marrow-resident+memory+T+cells+by+circulating,+antigen-bearing+dendritic+cells.">Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells.</a> Nat Immunol. 6, 1029-1037 (2005).</li><li>Mazo, I. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+is+a+major+reservoir+and+site+of+recruitment+for+central+memory+CD8++T+cells.">Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells.</a> Immunity. 22, 259-270 (2005).</li><li>Travlos, G. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+specialized+bone+marrow+endothelial+microdomains+for+tumour+engraftment.">In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.</a> Nature. 435, 969-973 (2005).</li><li>Cariappa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perisinusoidal+B+cells+in+the+bone+marrow+participate+in+T-independent+responses+to+blood-borne+microbes.">Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes.</a> Immunity. 23, 397-407 (2005).</li><li>Junt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Zoumi, A., Yeh, A., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence.">Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.</a> Proc Natl Acad Sci U S A. 99, 11014-11019 (2002).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs.">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annu Rev Immunol. 26, 585-626 (2008).</li><li>Yost, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+neutrophil+extracellular+trap+(NET)+formation:+a+novel+innate+immune+deficiency+of+human+neonates.">Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates.</a> Blood. 113, 6419-6427 (2009).</li><li>Devi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+imaging+reveals+a+new+leukocyte+recruitment+paradigm+in+the+glomerulus.">Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.</a> Nat Med. 19, 107-112 (2013).</li></ol>

The authors have nothing to disclose.

생체 내 영상화 방법의 중요한 점은 영상의 지속 시간을 최대화하고 염증 세포의 역학에 영향을 줄 수있는 세균 오염 및 염증의 위험을 최소화하기 위해 초점 안정성을 보장한다. 수술은 골수에 접근하는 것은 최소한의 수행으로 두개골 골수의 영상은 이러한 목표를 다음과 같습니다. 멸균 물질 및 방부제의 사용은 세포 항상성 교란을 유발할 수있는 감염의 위험을 제한하기 위해 필?...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

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			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:237px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ketamin</td>
			<td style="width:171px;">Merial</td>
			<td style="width:119px;"></td>
			<td>100mg/mL, anesthetic</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Xylazin</td>
			<td style="width:171px;">Bayer HealthCare</td>
			<td style="width:119px;"></td>
			<td>10mg/mL, anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Isofluran</td>
			<td style="width:171px;">Baxter</td>
			<td style="width:119px;"></td>
			<td>2.5%, anesthetic</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">O2/NO2</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>70/30 mixture, anesthetic</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:297px;">Rhodamin-Dextran</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>2MDa, 10mg/mL, Vascular staining</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:297px;">Ly6G-PE</td>
			<td style="width:171px;">Becton-Dickinson</td>
			<td style="width:119px;"></td>
			<td>clone 1A8, neutrophils staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Stereotactic holder</td>
			<td style="width:171px;">Home made</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Ethanol 70%</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>surgery</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sterile scissors and nippers</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Rubber ring</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td>18mm diameter, surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glubran 2</td>
			<td style="width:171px;">Queryo Medical</td>
			<td style="width:119px;"></td>
			<td>Surgical Glue, rubber ring fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Small gauge needles</td>
			<td style="width:171px;">Terumo</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss LSM 710 NLO multiphoton microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti:Sapphire crystal laser&#160;</td>
			<td>Coherent Chameleon Ultra</td>
			<td style="width:119px;"></td>
			<td>140fs pulses of NIR light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zen 2010</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Acquistion Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris Bitplane&#160;</td>
			<td style="width:171px;">Bitplane</td>
			<td style="width:119px;"></td>
			<td>Analysis Software, 3D automatic tracking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PBS 1X</td>
			<td style="width:171px;">D. Dutscher</td>
			<td style="width:119px;"></td>
			<td>surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Thermostated chamber</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>intravital imaging</td>
		</tr>
	</tbody>
</table>

tracking mouse bone marrow monocytes in vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

마우스 두개골 구조는 생체 내에 영상에 의해 골수 생리학을 연구 할 수있는 좋은 기회를 제공합니다. 프런트 정수리 주변 얇게 뼈, 그것은 뼈의 마모없이 medullar 틈새에 접근 할 수있다. 1 MacBlue 트랜스 제닉 마우스의 두개골 넓은 2D 필드를 나타내는 도표. 뼈 매트릭스는 주로 SHG (19)에 의해 쉽게 검출 I 콜라겐으로 구성되어있다. rhodamin - 덱스 트란의 주입은 골수?...

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

마우스 골수 단핵구를 추적<em&gt; 생체</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.4K 조회수.  Sorbonne Universités, UPMC Univ Paris 06, CR7. Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism. 

비디오: Tracking Mouse Bone Marrow Monocytes In Vivo

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

추적 연구 Qdot Nanocrystals와 Murine 골수 파생 돌기 세포의 생성 및 라벨링

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

연구

면역학 및 감염병학

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

분리, 정제 및 기능 연구 및 입양 전송 실험 쥐의 골수 호중구의 라벨링

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

뼈 대식 세포 생산에게 골수 유래

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

쥐 뒷다리 긴 뼈 해부 및 골수 격리

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

대한 대퇴골 창 회의소 모델<em&gt; 생체</em쥐의 골수에서&gt; 셀 추적

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

마우스 골수에서 형질 수지상 세포의 정화를위한 형광 활성화 셀 정렬

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

식별 및 Oligopotent 및 계보 커밋된 골수성 창시자 마우스 골 수에서 격리

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Vivo에서 마우스 모델을 측정 순진한 CD4 T 세포 활성화, 확산 및 골 수 유래 수지상 세포에 의해 유도 된 Th1 차별화

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...