건강 Kirchstein 원정대 predoctoral 교제 AI - 64128 (MPM), GM - 41514 (MDC), GM - 48071 (IP)의 국립 연구소

1) 쥐 한마리에서 모두 대퇴골의 뼈를 제거 <ol><li> 근육 버려야하고 (무릎 및 고관절) 이상과 관절 아래의 대퇴골 뼈를 노출 해부 가위를 사용합니다. 해부의 족집게로 대퇴골의 중심을 파악하고 최대한 그대로 epiphysis의를 떠나 위의와 관절 아래 컷. </li><li> 작은 해부 가위를 사용하여 가능한 한 많은 근육으로 닦아. RPMI 한 접시에 몽둥이를 전송합니다. </li><li> 모든 절차는 무균 미디어, 악기, 피펫 팁 문화 요리를 사용하여,이 시점부터 후드에서 수행되어야합니다. </li></ol> 2) 대퇴골의 뼈를 소독 : <ol><li> 후드에서 RPMI에서 얼음에 70 % 에탄올로 가득 작은 문화 요리에 유골​​을 전송할 수 있습니다. </li><li> 뼈가 얼음에 5 분 에탄올에 담그다 수 있습니다. </li></ol> 3) 무균 조건 하에서 골수를 수집합니다 <ol><li> 무균 여과 기본 DC 문화 매체를 포함하는 작은 문화 요리에 유골​​을 전송합니다. </li><li> 멸균 핀셋으로 폐기 접 관을 통해 뼈를 보유하고 멸균 가위로 (뼈의 종료) 각 epiphesis 잘라. 절단은 뼈의 중앙에 밝은 빨간색으로 골수를 노출해야합니다. </li><li> 멸균 주사기 (26-28 게이지 바늘)으로 살균 미디어 100-200에 대해 ML을 빨아. </li><li> 무균 미디어 신선한 음식을 통해 멸균 핀셋으로 뼈를 잡고 뼈의 한쪽에 바늘을 삽입합니다. 뼈 상단의 바늘의 끝 위치를 천천히 멸균 미디어 뼈를 씻으십시오. 당신이 뼈의 중심에있다면, 바늘은 쉽게 슬라이드한다. </li><li> 골수는 작은 조각 또는 한 조각으로 역시 밖으로 씻는다. 그것은 뼈 및 살균 미디어의 그릇에 밖으로 플러시해야합니다. </li><li> 완전히 뼈의 골수를 씻어 필요에 따라이 단계를 반복합니다. </li><li> 뼈를 깨끗하게되면, 그것은 흰색과 반투명 것입니다. </li><li> 다른 대퇴골 뼈이 절차를 반복합니다. 깨끗하게되면 각각의 대퇴부 뼈를 폐기하십시오. </li></ol> 4) 다시 일시 중지 및 골수 세포를 스핀 다운 : <ol><li> 멸균 팰컨 튜브에 세포를 전송합니다. </li><li> 골수이 아직도 그대로있다면, 아주 부드럽게 단일 세포 현탁액으로 골수를 깨고, 접시에 미디어를 피펫 아래. 이것은 몇 분 정도 걸릴 수 있으며 깎아지른듯한 강제로 세포를 죽이는 않도록 천천히 이루어져야합니다. </li><li> 어떤 다른 포유 동물 세포처럼 세포 원심 분리기. </li></ol> 5) 셀 용해 : <ol><li> 회전이 완료 후 원심 분리기에서 튜브를 제거하고 후드에있는 작은 쓰레기 (50 ML 팔콘 튜브)로 미디어에서 (가만히 따르다)를 부어. </li><li> 부드럽게 튜브의 바닥을 flicking하여 Resuspend 펠릿. </li><li> 무균 여과 물 10X DPBS 또는 10X PBS를 사용하여 다음 볼륨을 사용하여 적혈구 (RBCs)를 제거하는 물 용해를 수행 : <ul><li> 900 ML 무균 여과 물 </li><li> 100 ML 10X DPBS 또는 10X PBS </li></ul></li><li> 물을 추가한 후 10X PBS 50-10초 100 ML을 추가합니다. 그것은 즉시만을 RBCs가 lysed 것을 이렇게 매우 중요합니다. 그것은 두 pipettes 준비 채워 가지고하는 것이 좋습니다. </li><li> 무균 DC 기본 미디어 10 ML - 5 ML을 추가합니다. </li></ol> 6) 셀 카운트 : <ol><li> 후드에서 부드럽게 약간 역전, 45도 기울기 (미디어 튜브의 상단을 접촉하지 않도록)와 튜브를 소용돌이 치는하여 골수 세포의 6-11 ML를 섞어 </li><li> 계산을위한 멸균 튜브에 미디어 10 ML를 제거합니다. </li><li> 다시 모자 세포 원심 다시. </li></ol> 7) 도금 돌기 세포 : <ol><li> 전지는 1 X 106/mL의 밀도에 도금해야합니다. </li><li> 도금에 대한 기본 DC 미디어의 해당 금액의 원심 분리기에서 세포, 가만히 따르다 미디어와 다시 중단 세포를 제거합니다. </li><li> 조직 문화의 인큐베이터에 놓습니다. </li></ol> 8) 문화 케어 및 성숙 : 항상 더 많은 미디어를 추가하거나 실험에 사용하기 전에 문화를 확인하십시오. 세포의 일반적인 건강을 확인하고 잠재적인 오염을 찾습니다. DAY 0 : 돌기 세포 도금. DAY 3 : 미디어 비 adherant 세포의 75 %를 제거하고 다시 기본 DC 미디어를 추가합니다. DAY 6 : 초기 셀 도금 후 다음 절차를 사용하여 다시 판 전지 : <ol><li> (당신은 자기편 세포가 건조하고 싶지 않아요) 매우 약간 서부 유럽 표준시 번호판을 떠나, 살균 50 ML 팔콘 튜브의 일부가 아닌 adherant이 시점에서 DC가, 장소를 포함한다 미디어를 제거 </li><li> 각 플레이트에 PBS 3 MM EDTA (에틸렌 다이아 민 테트라 초산)을 추가하고 5 분 앉아 수 있습니다. </li><li> 45도 각도에서 접시를 잡고 부드럽게 부드럽게 비 adherant 세포를 이동시키다하는 접시의 바닥에 대한 미디어를 피펫 아래. </li><li> EDTA (에틸렌 다이아 민 테트라 초산) / PBS는 두껍게 나타내는 세포의 바닥에서 수집되는 되야지판. </li><li> 이 몇 분 후에 세포 혼합물은 50 ML 팔콘 튜브로 전송하실 수 있습니다. </li><li> 각 플레이트에 대해이 절차를 수행한 후 모든 셀을 스핀. </li><li> 1 밀도에 카운트 및 플레이트 세포 X 10 ^ 새로운 살균 10cm 문화 요리 보조 DC 미디어 6 회 판. 조직 문화의 인큐베이터로 세포를 반환합니다. </li></ol> DAY 10-11 자극 / 항원 로딩 준비 성숙 DC가. 9) 돌기 세포 자극 : <ol><li> 활성화 및 펩티드 로딩을​​ 위해 원하는 펩타이드와 함께 LPS를 (lipopolyscaccharide)를 사용합니다. LPS의 소스에 따라 서로 다른 자극 조건은 더 나은 결과를 제공할 수 있습니다. 또한, DC 프레 젠 테이션을 위해 펩타이드의 원하는 금액을 최적화해야합니다. 이 프로토콜의 자극 조건은 10​​0 NG / ML LPS 100 UG / ML OVA (ovalbumin)되었습니다. </li><li> 로드 항원 / 사전 수확 18-24시간에 대한 LPS와 치료 / 사용합니다. </li></ol> 돌기 세포 미디어 : 모든 후 무균 필터가 추가되었습니다 다른 돌기 세포 배양 조건을 검토하고 그 결과 돌기 세포 표현형에 대한 토론 섹션을 참조하십시오. 이러한 문화 조건이 성숙 돌기 세포를 생산하도록 설계되는 급속 18~24시간 피하 주사하고 효율적으로 현재의 항원 후 배수 림프절에 집. 기본 DC 미디어 : 모든 후 무균 필터가 추가되었습니다 <ul><li> 500 ML IMDM (500mL 최종 볼륨 55-60 ML 제거) </li><li> 50 ML 열 inactivated FBS </li><li> 200 MM L - Gln, 2 mm의 최종 농도 5 ML </li><li> 100 IU / ML 페니실린과 100 MG / ML 스트렙토 마이신 (10,000 IU / ML 펜 및 만 MG / ML Strep 주식 5 ML) </li><li> 50 음 B - 나 (14.3 M B - 내 물건은 화학 후드에 1:100을 희석, 50 micromolar 집중력 100 ML 미디어 당 35 UL 사용) </li><li> 20-30 NG / ML GM - CSF </li><li> 100-400 IU / ML IL - 4 약 10-40 NG / ML </li></ul> 성숙의 *에 대한 SecondaryDC 미디어 100 NG / ML TNF - 알파 (기본 DC 미디어에 추가) * 참고 : GM - CSF + IL - 4는 어려서 돌기 세포를 생산한다. GM - CSF를 사용하여 + IL4 (400 IU / UL) + TNF - 알파 (100 UG / ML)은 성숙 단핵구 파생 돌기 세포 유사 세포를 생성합니다. 중년 여인 돌기 세포는 미숙 돌기 세포와 문화가 내생 TNF - stromal 세포에서 출시 4 수 있습니다 한한 효율적으로 항원을 차지하지 알려져 있습니다.

<ol>
	<li>Miller, M. J., Safrina, O., 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=Imaging+the+single+cell+dynamics+of+CD4++T+cell+activation+by+dendritic+cells+in+lymph+nodes.">Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes.</a> J Exp Med. 200, 847-856 (2004).</li><li>Henrickson, S. E., 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=T+cell+sensing+of+antigen+dose+governs+interactive+behavior+with+dendritic+cells+and+sets+a+threshold+for+T+cell+activation.">T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation.</a> Nat Immunol. 9, 282-291 (2008).</li><li>Jefford, 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=Functional+comparison+of+DCs+generated+in+vivo+with+Flt3+ligand+or+in+vitro+from+blood+monocytes:+differential+regulation+of+function+by+specific+classes+of+physiologic+stimuli.">Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli.</a> Blood. 102, 1753-1763 (2003).</li><li>Inaba, 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=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 176, 1693-1702 (1992).</li><li>Lutz, M. 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=Differential+functions+of+IL-4+receptor+types+I+and+II+for+dendritic+cell+maturation+and+IL-12+production+and+their+dependency+on+GM-CSF.">Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF.</a> J Immunol. 169, 3574-3580 (2002).</li><li>Xu, Y., Zhan, Y., Lew, A. M., Naik, S. H., Kershaw, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+development+of+murine+dendritic+cells+by+GM-CSF+versus+Flt3+ligand+has+implications+for+inflammation+and+trafficking.">Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.</a> J Immunol. 179, 7577-7584 (2007).</li><li>Inaba, K., Swiggard, W. J., Steinman, R. M., Romani, N., Schuler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+dendritic+cells.">Isolation of dendritic cells.</a> Curr Protoc Immunol. 3, (2001).</li><li>Winzler, 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=Maturation+stages+of+mouse+dendritic+cells+in+growth+factor-dependent+long-term+cultures.">Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.</a> J Exp Med. 185, 317-328 (1997).</li><li>Koch, F., 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=Tumor+necrosis+factor+alpha+maintains+the+viability+of+murine+epidermal+Langerhans+cells+in+culture,+but+in+contrast+to+granulocyte/macrophage+colony-stimulating+factor,+without+inducing+their+functional+maturation.">Tumor necrosis factor alpha maintains the viability of murine epidermal Langerhans cells in culture, but in contrast to granulocyte/macrophage colony-stimulating factor, without inducing their functional maturation.</a> J Exp Med. 171, 159-171 (1990).</li><li>Miller, M. J., Hejazi, A. S., Wei, S. H., 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=T+cell+repertoire+scanning+is+promoted+by+dynamic+dendritic+cell+behavior+and+random+T+cell+motility+in+the+lymph+node.">T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node.</a> Proc Natl Acad Sci U S A. 101, 998-1003 (2004).</li><li>Wei, S. H., 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=Ca2++signals+in+CD4++T+cells+during+early+contacts+with+antigen-bearing+dendritic+cells+in+lymph+node.">Ca2+ signals in CD4+ T cells during early contacts with antigen-bearing dendritic cells in lymph node.</a> J Immunol. 179, 1586-1594 (2007).</li><li>Castellino, F., 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=Chemokines+enhance+immunity+by+guiding+naive+CD8++T+cells+to+sites+of+CD4++T+cell-dendritic+cell+interaction.">Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction.</a> Nature. 440, 890-895 (2006).</li></ol>

돌기 세포는 적응 면역 반응과 날짜에 특징이 가장 효율적인 항원 제시 세포의 핵심 중재자입니다. 문학 인간 및 마우스의 돌기 세포 배양을위한 방법은 다른 돌기 세포 유형의 발전에 영향을 미치는 데 사용 크린 시토킨의 종류에 차이가 있습니다. 특히, GM - CSF, flt3L, IL4, IL13, TNF - 알파와 IFN - 감마는 성숙 미숙, 염증 및 정상 상태 체외 3-7에서 돌기 세포를 파생 murine 골수와 같은 생산하는 다른 조합에 사...

generation of bone marrow derived murine dendritic cells for use in 2-photon imaging

murine 돌기 세포의 준비를 위해 여러 가지 방법이 문헌에서 찾을 수 있습니다. 여기, 우리는 문화에 이상 85% CD11c 높은 돌기 세포를 생산하는 방법을 제시 것을 피하 주입 및 항원 구체적인 T 세포 (동영상 참조) 현재 항원 후 배수 림프절의 고향. 또한, 우리는 일 10시 교양 세포의 형태가 성숙 돌기 세포와 세포의 &lt;85% CD11chigh 아르의 전형이며, 돌기 세포 성숙을 추적하기 위해 에센 악기에게 Incucyte을 사용합니다. 2 - 광자 이미징에 의해 주변 림프절에 항원 프레 젠 테이션의 연구는 돌기 세포와 T 세포 상호 작용 1, 2의 세 개의 서로 다른 단계가있다는 것을 밝혔다. 페이즈 I는 매우 운동성이있는 특정 항원 T 세포와 돌기 세포 1, 2를 들고 항원 사이의 간단한 시리얼 연락처로 구성되어 있습니다. 차 두 항원에 특정한 T 세포와 돌기 세포 1, 2 베어링 항원 사이에 장시간 접촉에 의해 표시됩니다. 마지막으로, 단계 III는 운동성을 회복하고 1, 2,을 분할하기 시작, 돌기 세포에서 분리 T 세포에 의해 특징입니다. 이것은 항원 -로드 셀 추적 염료 분류 돌기 세포의 2 광자 영상으로 분석할 수 항원 특정 상호 작용의 유형의 한 예입니다.

Watch this Scientific Journal Video about Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging at JoVE.com

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

돌기 세포에 의해 이차 림프​​ 장기의 항원 프레 젠 테이션은 T 세포 적응 면역 반응을 중재의 개시를 위해 중요합니다. 여기서 우리는 골수의 문화 파생 murine 돌기 세포, 활성화 및 2 광자 이미징에 대한 라벨을 보여줍니다.

골수의 생성은 2 광자 영상에서 사용하기 위해 Murine 돌기 세포를 유래

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

generation-bone-marrow-derived-murine-dendritic-cells-for-use-2

Research

JoVE Journal

Biology

31.0K 조회수.  University of California, Irvine (UCI). 돌기 세포에 의해 이차 림프​​ 장기의 항원 프레 젠 테이션은 T 세포 적응 면역 반응을 중재의 개시를 위해 중요합니다. 여기서 우리는 골수의 문화 파생 murine 돌기 세포, 활성화 및 2 광자 이미징에 대한 라벨을 보여줍니다.

비디오: Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

해부와 마우스에서 주변 림프절의 2 광자 이미징

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

연구

생물학

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

2 광자 현미경을 사용하여 마우스 Thymus의 현장 영상에서

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

두개골의 창문을 통해 네크로 텍스의 혈류의 2 광자 이미징을위한 방법

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

레이어의 VIVO에서 2 광자 칼슘 이미징 마우스의 2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

골수 엽 성의 전구 물질에서 골수양 돌기 세포의 문화

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

골수에 조혈 세포의 유도

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

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) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

마우스 골수에서 파골 세포의 유도

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Murine 골 수 유래 수지상 세포와 대 식 세포에 형광 성 융해 단백질의 표정

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...