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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+roles+of+monocyte+subsets+in+steady+state+and+inflammation.">The multiple roles of monocyte subsets in steady state and inflammation.</a> Cell Mol Life Sci. 67, 2685-2693 (2010).</li><li>Shi, 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=Bone+marrow+mesenchymal+stem+and+progenitor+cells+induce+monocyte+emigration+in+response+to+circulating+toll-like+receptor+ligands.">Bone marrow mesenchymal stem and progenitor cells induce monocyte emigration in response to circulating toll-like receptor ligands.</a> Immunity. 34, 590-601 (2011).</li><li>Jacquelin, 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=CX3CR1+reduces+Ly6Chigh-monocyte+motility+within+and+release+from+the+bone+marrow+after+chemotherapy+in+mice.">CX3CR1 reduces Ly6Chigh-monocyte motility within and release from the bone marrow after chemotherapy in mice.</a> Blood. 122, 674-683 (2013).</li><li>Swirski, F. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+chemokines+and+chemokine+receptors+in+inflammation.">The many roles of chemokines and chemokine receptors in inflammation.</a> N Engl J Med. 354, 610-621 (2006).</li><li>Milo, I., 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+imaging+reveals+promiscuous+crosspresentation+of+blood-borne+antigens+to+naive+CD8++T+cells+in+the+bone+marrow.">Dynamic imaging reveals promiscuous crosspresentation of blood-borne antigens to naive CD8+ T cells in the bone marrow.</a> 122, 193-208 (2013).</li><li>Cavanagh, L. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+structure,+function,+and+histology+of+the+bone+marrow.">Normal structure, function, and histology of the bone marrow.</a> Toxicol Pathol. 34, 548-565 (2006).</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=Hematopoietic+progenitor+cell+rolling+in+bone+marrow+microvessels:+parallel+contributions+by+endothelial+selectins+and+vascular+cell+adhesion+molecule+1.">Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.</a> J Exp Med. 188, 465-474 (1998).</li><li>Rashidi, N. M., 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+time-lapse+imaging+of+mouse+bone+marrow+reveals+differential+niche+engagement+by+quiescent+and+naturally+activated+hematopoietic+stem+cells.">In vivo time-lapse imaging of mouse bone marrow reveals differential niche engagement by quiescent and naturally activated hematopoietic stem cells.</a> Blood. , (2014).</li><li>Sipkins, 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=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.

<table><tbody><tr><td>Ketamin</td><td>Merial</td><td></td><td>100 mg/ml, anesthetic</td></tr><tr><td>Xylazin</td><td>Bayer HealthCare</td><td></td><td>10 mg/ml, anesthetic</td></tr><tr><td>Isofluran</td><td>Baxter</td><td></td><td>2.5%, anesthetic</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;/NO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>70/30 mixture, anesthetic</td></tr><tr><td>Rhodamin-Dextran</td><td>Invitrogen</td><td></td><td>2 MDa, 10 mg/ml, Vascular staining</td></tr><tr><td>Ly6G-PE</td><td>Becton-Dickinson</td><td></td><td>clone 1A8, neutrophils staining</td></tr><tr><td>Stereotactic holder</td><td>Home made</td><td></td><td>surgery</td></tr><tr><td>Ethanol 70%</td><td></td><td></td><td>surgery</td></tr><tr><td>Sterile scissors and nippers</td><td></td><td></td><td>surgery</td></tr><tr><td>Rubber ring</td><td></td><td></td><td>18 mm diameter, surgery</td></tr><tr><td>Glubran 2</td><td>Queryo Medical</td><td></td><td>Surgical Glue, rubber ring fixation</td></tr><tr><td>Small gauge needles</td><td>Terumo</td><td></td><td>surgery</td></tr><tr><td>Zeiss LSM 710 NLO multiphoton microscope&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td>Microscope</td></tr><tr><td>Ti:Sapphire crystal laser&amp;nbsp;</td><td>Coherent Chameleon Ultra</td><td></td><td>140 fsec pulses of NIR light</td></tr><tr><td>Zen 2010</td><td>Carl Zeiss</td><td></td><td>Acquistion Software</td></tr><tr><td>Imaris Bitplane&amp;nbsp;</td><td>Bitplane</td><td></td><td>Analysis Software, 3D automatic tracking</td></tr><tr><td>PBS 1X</td><td>D. Dutscher</td><td></td><td>surgery</td></tr><tr><td>Thermostated chamber</td><td>Carl Zeiss</td><td></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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.5K 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

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 ...

연구

면역학 및 감염병학

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 ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

라이브 쥐의 대퇴부 골수 흡인

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

의학

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

실시간으로 마취 마우스에 생체 내에 현미경으로 장간막 혈관에서 호중구와 단핵구 이미징

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

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, ...

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.

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 ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

호중구 계보 세포의 질량 세포 분석 분석을위한 전체 골수의 준비

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

생물학

<meta charset="utf8"></head><body>살아있는 쥐의 골수 연구를 위한 대퇴 내 주사

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>살아있는 마우스를 사용한 간소화된 대퇴강 주사로 지속적인 골수 분석 가능

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

살아있는 마우스를 사용한 간소화된 대퇴강 주사로 지속적인 골수 분석 가능

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

마우스 골수 단핵구를 추적

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

분리 및 설치류 골수의 정맥 주사 유래 단핵구

라이브 쥐의 대퇴부 골수 흡인

실시간으로 마취 마우스에 생체 내에 현미경으로 장간막 혈관에서 호중구와 단핵구 이미징

대한 대퇴골 창 회의소 모델

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

호중구 계보 세포의 질량 세포 분석 분석을위한 전체 골수의 준비

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

살아있는 마우스를 사용한 간소화된 대퇴강 주사로 지속적인 골수 분석 가능

살아있는 마우스를 사용한 간소화된 대퇴강 주사로 지속적인 골수 분석 가능