이 프로토콜은 테스트 데이빗 스캐든의 연구실에 최적화되었습니다. 기금은 다나 파버 암 연구소 / 매사 추세츠 종합 병원 임상 혈액 및 종양학 원정대 프로그램에 의해 제공됩니다.

<ol><li> 우리는 실험에 사용되는 세포를 추출하여 유도 실험을 시작합니다. 받는 동물의 긴 뼈와의 틈새로 전체 골수의 위치 추적 이러한 C57BL/6J (WT)로 WT 동물에서 추출한 골수 세포에 대해 서로 다른 있으며, 유전자 변형되는 이러한 경우이 현재 실험을 위해 우리는 밖으로 찾는 데 관심이있다 Lysophosphatidic 수용체 1 (LPAR1) (KO)에 knockouts. </li><li> 우리가 실험을 시작하기 전에, 우리는 우리가이 실험에 필요한 자료를 수집합니다. 세포 대신 전 생체내 분석에 사용되는받는 동물에 이식 될 특히 우리가 실험실에서 모든 마우스 해부는 층류 조직 문화 후드에서 수행됩니다. 우리는 후드 안쪽에 파란색 시트를 삽입하여 시작합니다. 필요한 다른 개체는 포셉 한 켤레와 autoclaved되었습니다 가위 날카로운 쌍, 살균 일회용 메스와 알코올 면봉을 포함합니다. 우리는 수정 PBS 및 살균 6 자 번호판, 절구가 필요합니다, 유봉, 50cc 청색 원뿔 관과 70uM 일회용 필터를 얹은. </li><li> 우리가 하나 WT 한 KO 마우스를 복용하여 시작합니다. </li><li> 이 생쥐는 CO 2 흡입에 의해 euthanized 후 5 초 동안 isopropranolol 70%에 담근 수 있습니다. 이 단계는 마우스 모피와 우리 손에 관심 세포의 오염이 셀이나 실험 결과의 수율에 타협을 주도하지 않은 것을 방지할 수 있습니다. </li><li> 생쥐는 다음 포셉와 날카로운 가위를 사용하여 차례로 해부하고 있습니다. 우리는 일반적으로 일상의 문제로 WT 마우스로 시작합니다. 작은 싹둑는 복부와 피부가 다음 손을 사용 늘어입니다 overlying 마우스의 복부 피부에 이루어집니다. 피부는 쉽게 뒷다리 사지에서 온다. 대퇴골과 경골은 다음이가 골수의 대량 포함되어 대퇴골의 머리를 이동시키다하지 돌보는 압축이 풀립니다. 위쪽 팔다리는 몸통이 고정되어 유지하면서 단순히 그들에게 견인을 적용하여 동물의 몸통에서 분리됩니다. 이것은 한 조각으로 상부 사지를 제거합니다. humeri 그런 다음 상단 사지에서 추출됩니다. </li><li> 삭제 뼈가 여섯 잘 접시에 추가되었습니다 수정하는 PBS에 배치됩니다. 수정 PBS는 열 inactivated 태아 소 혈청과 EDTA (에틸렌 다이아 민 테트라 초산) 0.5 M pH8 2 CC 2 ML를 추가하여 이루어집니다. 이 솔루션은 다음 Nalgene 0.45 음 .. 필터를 통해 여과되고 최소한 한 달 동안 섭씨 4 도에 저장할 수 있습니다. </li><li> 그것은 정확하게 WT와 KO 뼈를 식별하기 위하여 접시에있는 우물에 레이블을하는 것이 필수적입니다. </li><li> 마우스 주검이 제거되고 해제 처리됩니다. </li><li> 새로운 시트는 후드에 배치되고 뼈는 일회용 메스를 사용하여 청소합니다. </li><li> 청소 뼈가 절구에 한 번에 하나의 마우스를 위치하고 있습니다. 수정 PBS의 두 ML은 절구에 추가되고 뼈는 유봉을 사용하여 조각하고 있습니다. 그것은 10과 함께 다음과 유봉의 서있는 움직임과 뼈가 원형 회전 움직임으로 그들을 격파 첫번째 조각하는 것이 중요합니다. 난 보통 시계 방향으로 50, 50 다음 시계 반대 방향으로 운동을하고 그래서 절구에 남아있는 자료는 색상과 수정된 PBS에 흰색 때까지​​ 계속 흰색 또는 투명 남아있는 자료에 추가됩니다. </li><li> 50 원형 파쇄 움직임의 모든 설정하면, 우리는 박격포에서 50 ML 원뿔 튜브의 상단에 위치 70 음 필터 액체를 전송합니다. 필터마다 전송 후 박격포에 PBS 2 ML을 추가하는 것을 잊지 마십시오. 당신은이 세포의 apoptosis에 이끌로 시도하고 격파 마른 뼈를 또는 시간의 기간 동안 그들이 건조두고 싶지 않아. </li><li> 절구에 자료가 흰색이됩니다되면, 필터의 재질은 박격포로 전송되고 분쇄 다시 수행됩니다. 이것은 필터에있는 세포가 낭비되지 않도록 보장합니다. 보통 그것은 다시 세포의 추출이 완료되는 시점 색상의 박격포 흰색의 소재를 만들기 위해 원형 회전 운동 중 하나 또는 두 세트 걸립니다. </li><li> 원뿔 튜브의 솔루션은 이제 (주십시오 WT와 KO 마우스 별도의 세포를 유지하는 기억 해요. 따라서 당신이 10 1,200 rpm으로 두 개의 다른 튜브 (KO 세포와 WT 세포와 함께 한 다른). 원심 분리기를해야합니다 centrifuged입니다 분. </li><li> 각각의 튜브에 대해 별도의 흡인기의 pipettes를 사용하여 표면에 뜨는를 대기음. 각각의 펠렛에 ACK 용해 버퍼 2 참조를 추가합니다. 이 용해 버퍼 멸균해야합니다. 5 분 얼음에 품어. </li><li> 각각의 튜브에 수정된 PBS 8 CC를 추가합니다. 96 잘 접시에 두 개의 별도 우물로 10 각 튜브의 UL과 피펫을 가져가라. 원심 분리기에 튜브를 삽입하고 10 분 동안 1,200 rpm으로 그들을 스핀. </li><li> 튜브가 회전하는 동안 관심의 세포를 지니고 있던 96 잘 접시의 각각 잘하는 tryphan 청색 색소의 30 UL 및 수정된 PBS 60 UL를 추가합니다. </li><li> hemocytometer이 혼합 10 UL (최대 아래로 pipetting으로 잘 섞어주세요) 추가하고 네 가지 바깥 광장에있는 세포를 계산합니다. </li><li> ML 당 각 샘플의 셀 개수는 4 multiplie로 나눈 따라서 계산 세포의 숫자입니다100 000에 의해 D. </li><li> 원심 분리가 완료되면, 원심에서 튜브를 제거하고 ML 당 20,000,000 세포의 농도에서 칼슘, 마그네슘 또는 L의 글루타민없이 PBS에 세포를 resuspend. 당신은 가능성 KO 동물에 대한 WT 볼륨 5 ML에 대한 볼륨 약 5 ML을 것입니다. 이 단계 이후에 우리는 프로토콜 PBS를 수정하지 PBS를 사용합니다. </li><li> 각각의 튜브를 추가하려면 DiI, 세포 얼룩의 염료는 5uM의 최종 농도를 얻을 수 있습니다. 10 초 즉시 와동. 10 분 37도에서 부화하고 불빛에 노출하지 마십시오. </li><li> 1,200 rpm으로 10 분간 PBS와 스핀으로 튜브를 입력합니다. </li><li> 원심 분리 후, 뜨는을 기음과 ML 당 40,000,000 세포에 PBS에 세포를 resuspend. </li><li> 27G 바늘 (WT 마우스 및 KO 마우스 5 주사기 5 주사기) 1 CC의 주사기로 500 UL을로드합니다. 그것은 각 코호트 6 주사기를 준비하는 신중한 수 있습니다. 우리가 다음에 할 건데 꼬리 정맥 주사가 힘들 수 있으므로 여분의 하나가 도울 수 있습니까. </li><li> 험악에서 DiI을 유지하기 위해 호일로 주사기를 커버. </li><li> 각받는 동물로 세포 혼합물 500 UL을 주사, (각 코호트 5). </li><li> 이 동물은 4-24시간 전에 분사에 방사선 조사의 9 쥐와 함께 조사해야합니다. </li><li> 주사 후 48 시간, 각받는 동물의 뒷다리 사지에서 수확 골수. </li><li> 기증자에 대한 위에 설명된대로 처리합니다. PBS 1 CC의 각 동물의 세포를 Resuspend. hemacytometer를 사용하여 셀 개수를 구합니다. </li><li> BD에서 LSR II 흐름 cytometer 또는 이에 상응하는 악기를 읽어 보시기 바랍니다. 결과는 DiI 긍정하는 분석 골수의 비율이나 세포의 절대 숫자로 표현할 수있다. </li></ol>

<ol>
	<li>Adams, G. 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=Stem+cell+engraftment+at+the+endosteal+niche+is+specified+by+the+calcium-sensing+receptor.">Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.</a> Nature. 439 (7076), 599-603 (2006).</li><li>Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+hematopoiesis.">Chemokines in hematopoiesis.</a> Curr Opin Hematol. 15 (1), 49-58 (2008).</li></ol>

호밍는 과정입니다 의하여 줄기 세포, 전구 세포와 차별화된 세포, 혈액 스트림 또는 마우스의 골수 캐비티에 주입 후 골수에 그들의 방법을 포함하여 골수 세포. 정의에서 분명로서, 과학자가 조혈 줄기 세포, 전구 세포 또는 immunophenotyping으로 식별 및 세포 분류에 의해 격리 차별화된 세포의 위치 추적을 공부하기로 수도 있습니다. 일반적으로 과학자들은 혈통 마커에 대한 고갈 골수 세포를 주?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>DPBS (Dulbecco&#x2019;s Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Mediatech, Inc</td>
<td>21-031-CV</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Valley Biomedical, Inc</td>
<td>BS3033</td>
<td>&nbsp;</td>
<tr>
<td>0.5M EDTA (pH 8.0)</td>
<td>&nbsp;</td>
<td>Boston BioProducts</td>
<td>BM-150</td>
<td>&nbsp;</td>
<tr>
<td>Tryphan Blue solution</td>
<td>&nbsp;</td>
<td>Mediatech, Inc</td>
<td>25-900-CI</td>
<td>&nbsp;</td>
<tr>
<td>ACK Lysing Buffer</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>10-548E</td>
<td>&nbsp;</td>
<tr>
<td>Isopropranolol U.S.P.</td>
<td>&nbsp;</td>
<td>Denison Pharmaceuticals, Inc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vybrant DiD cell-labelling solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>V22887</td>
<td>&nbsp;</td>
<tr>
<td>Vybrant DiI cell-labelling solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>V22885</td>
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homing of hematopoietic cells to the bone marrow

호밍는 이식 조혈 세포와 합치다 여행이나 골수에 거주를 설정하실 수 있습니다 약자 현상입니다. 다양한 chemomkines과 수용체는 조혈 줄기 세포의 유도에 관여하고 있습니다. [1, 2] 본 논문은 조혈 줄기 세포 연구에 사용되는 고전적인 추적 프로토콜을 설명합니다. 일반적으로 이것은 누구의 추적 조사 요구되는 세포 인구를 분리 관심 염색이 인구가 얼룩과받는 동물의 혈류에 이들 세포를 주입이 포함됩니다. 받는 동물은 다음 주입 및 비율이나 관심의 염색에 대한 양성 세포의 절대적인 번호에 대한 평가 골수 후 미리 정해진 시간에 희생됩니다. 가장 일반적인 실험 계획, 두 유전자 별개의 동물로부터 조혈 세포 (야생 동물 유형 및 해당 노크 아웃)의 위치 추적 효율 중 하나에 비교됩니다. 이 문서는 실험과 같은 내용의 프레임 워크에있는 조혈 세포 유도 프로토콜을 설명합니다.

Watch this Scientific Journal Video about Homing of Hematopoietic Cells to the Bone Marrow at JoVE.com

Homing of Hematopoietic Cells to the Bone Marrow

이 문서는 골수에서 틈새에 조혈 세포의 추적을 연구하는 데 사용하는 프로토콜을 설명합니다.

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 ...

homing-of-hematopoietic-cells-to-the-bone-marrow

Research

JoVE Journal

Biology

29.4K 조회수.  MGH - Massachusetts General Hospital. 이 문서는 골수에서 틈새에 조혈 세포의 추적을 연구하는 데 사용하는 프로토콜을 설명합니다.

비디오: Homing of Hematopoietic Cells to the Bone Marrow

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

태반에서 조혈 줄기 세포의 분리 및 분석

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

연구

생물학

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 ...

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

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% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

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% ...

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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral - 중재 최저 동안 예 VIVO</em인간 조혈 줄기 세포의> 적혈구 조혈

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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 ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

쥐 골 골에서의 파 골 세포 전 구체의 분리, 정제 및 분화

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

마우스 신선한 골수 이별의 프로플라츠 형성 역학

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...