O.A.-W. is supported by an NIH K08 Clinical Investigator Award (1K08CA160647-01), a U.S. Department of Defense Postdoctoral Fellow Award in Bone Marrow Failure Research (W81XWH-12-1-0041), the Josie Roberston Investigator Program, and a Damon Runyon Clinical Investigator Award with Support from the Evan&#8217;s Foundation.

이 프로토콜에 설명 된 모든 동물의 절차는 실험 동물의 관리 및 사용에 대한 지침에 따라 실시하고, 메모 리얼 슬로안 - 케터링 암 센터의 기관 동물 케어 및 사용위원회 (IACUCs)에 의해 승인되었다. 대퇴골에서 골수의 1. 흡인 (BM) 세포 <ol><li> 정밀 기화기 투여 이소 플루 란 (1-4 %)과 대퇴 골수 흡인을 받게 될 것이다 마우스를 마취. 마취 심도가 수술 조작 및 / 또는 귀, 발가락, 테일 핀치와 관련된 호흡에 변화가없는 것을 관찰함으로써 절차 내내 매 50-10 분을 모니터해야한다. 마취는 이소 플루 란으로 유도하고 또한 이소 플루 란과 절차를 통해 유지된다. 0.05-0.1 밀리그램의 부 프레 노르 핀 투여 량의 선제 사전 절차 용량 / kg 피하 매 6-12 시간은 ADJ로 절차와 관련된 통증을 방지하는데 사용될 수있다Carprofen에 unct. </li><li> 각막 건조를 방지하기 위해 마취의 마우스 다음 유도의 눈 안과 연고를 적용합니다. </li><li> 모피는 신중하게 의도 열망 사이트의 면적보다 약 150 % 더 큰 피부의 영역에서 잘립니다. 느슨한 모피는 촉촉한 거즈 패드로 제거됩니다. 절차를 수행하는 동안 저체온증을 방지하기 위해 순환 온수 패드 또는 다른 안전한 온도 조절 장치로 통제 된 표면에서 마우스를 보관하십시오. </li><li> (포비돈 요오드 (베타 딘) 또는 클로르헥시딘 (Nolvasan) 스크럽 70 % 이소 프로필 알코올이나 70 % 에탄올을 적신 거즈 스폰지 하나와 교류) 교류 수술의 세 가지 세트로 포부를 받게 될 것이다 대퇴골을 포함하는 전체 다리를 소독. </li><li> BM을 흡입하기 전에 멸균 인산 완충 생리 식염수 (PBS)으로 :; : 0.5 ㎖ 투베르쿨린 주사기 (27.5 G 게이지 0.5 cc의 볼륨)을 적 십니다. PBS의 200 ~ 500 ㎕ 씩 주사기를 입력하고 즉시 PBS를 추방. 이 절차 2-3t를 반복IME를. </li><li> 반지 손가락 또는 다섯 번째 손가락 하나와 경골을 눌러 대퇴골에서 경골의 굴곡을 유지합니다. 적절한 마취 비행기가 달성되었는지 확인합니다. 주사기는 엄지와 집게 손가락을 사용하여 유지된다. 이는 관절 구가 노광 될 수 있도록하고 바늘의 삽입을 용이하게한다. </li><li> 바늘이 대퇴골 두 관절 구 사이에 단단히 박혀되도록 주사기를 적시 한 후, 슬개를 통해 바늘을 삽입합니다. 엄지 손가락과 집게 손가락으로 대퇴골 골단 부근 골간을 유지함으로써, 바늘이 쉽게 대퇴골의 샤프트에 삽입된다. </li><li> 이 대퇴골 축과 평행이되도​​록 외측으로 상향 바늘 스위블. 이 작업은 대퇴골 축에서 골수 내용의 검색을 용이하게한다. </li><li> 대퇴 골수 구멍으로 천천히 누르면서 시계 반대 방향으로 바늘을 시계 방향으로 돌려. 온화 싸이 이동하여 바늘의 정확한 위치 확인옆으로 ringe. </li><li> 앞뒤로 BM 캐비티 내에서 바늘을 이동하는 동안 부드럽게, 부정 압력을 생성, 다시 바늘 플런저를 잡아 당깁니다. 주 : 흡입 된 BM의 부피는 일반적으로 0.4-0.8 × 106 단핵 세포에 상당 약 5 μL 것이다. 성공 열망은 주사기의 기지에있는 바늘의 위쪽에있는 혈액의 모양을 시각적으로 확인 될 것이다. 피가 주사기에서 볼 경우 그것은 작은 뼈 또는 조직 조각이 바늘에 걸린 가능성이 높습니다. 이것은 (이것은 주사기 BM 흡입 전에 PBS로 채워져해야 하나의 이유이다) PBS에 상하 플런저를 이동함으로써 바늘로부터 제거 될 수있다. 조직이 주사기로부터 빠질 수없는 경우, 새로운 바늘과 주사기 (다시 PBS의 200-500 μL와 주사기를 젖은 상태) 사용. </li><li> BM이 성공적으로 대퇴골에서 흡입되면, 대퇴골 및 마우스에서 바늘과 주사기를 제거합니다. </li><li> 의 미세 튜브 홍보에 흡입 된 BM 이동PBS 500 μL와 efilled. 대부분의 응용 프로그램, BM 세포는 추가 처리를 가능하면 때까지 얼음에 보관해야합니다. </li><li> 절차의 완료 후, 5 ㎎ / ㎏ 피하 carprofen으로 진통을 관리 할 수​​ 있습니다. 그 후 마취에서 마우스를 제거하고 완전히 회복 될 때까지 가열 패드에 배치합니다. 참고 : 만약 제대로 흡입 절차에 따라 경험 합병증이나 고통이 없어야합니다. </li><li> 주택 지역에 마우스를 반환하기 전에, 그들은 음식과 물을 ambulate 및 도달 할 수 있는지. 다음 24 시간에 고통이나 감염 후 절차의 징후 쥐를 관찰합니다. 증상은 다음을 포함한다 : 지속적인 출혈, 빈혈, 혼수를. 이러한 징후는 포스트 절차를 보이는 경우, 동물 (들) 안락사한다. 주 : BM 흡입 / 샘플링을 반복 할 수 있지만 반복 절차는 동일 다리 반복 외상을 방지하기 위해 대향 대퇴골을 수행하여야한다. FR에 대한 가능한 약간의 정보가있다equency 그 BM 열망을 수행 할 수 있습니다. 대퇴 골수 흡입은 일반적으로 더 자주 매 2 주 이상 반복되지 않습니다. </li></ol> 으로 발음 BM 세포의 세포 내용의 2. 평가 <ol><li> 4 O를 C 또는 RT에서 5 분 300 XG에서 원심 분리하여 미세 원심 분리 튜브에 BM 열망과 장소에서 검색 펠렛 세포. </li><li> 뜨는을 대기음 후 ACK 적혈구 세포 용해 버퍼 ( &quot;암모늄, 염화 칼륨,&quot;용해 버퍼) 500 μL에 펠렛을 재현 탁. </li><li> 10 분 동안 적혈구 용해 완충액에 세포를 부화 후 PBS 1 ㎖를 추가하고 4 입출력 C 또는 RT에서 5 분 동안 300 XG에 다시 혼합물을 스핀 다운. 참고 : 적혈구 용해 펠릿 지금 BM 단핵 세포로 구성되어 있습니다. 이들은 FACS 염색, 세포 계수, 이식, cytospin 분석 및 / 또는 기타 사용 (희생 생쥐에서 수확 BM 세포를 사용하는 것처럼)에 재현 탁 할 수있다. </li></ol>

<ol>
	<li>Sundberg, R., Hodgson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspiration+of+bone+marrow+in+laboratory+animals.">Aspiration of bone marrow in laboratory animals.</a> Blood. 4, 557-561 (1949).</li><li>Schmitz, M., Bourquin, J. -. P., Bornhauser, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+technique+for+intrafemoral+injection+and+bone+marrow+sampling+in+mouse+transplant+models.">Alternative technique for intrafemoral injection and bone marrow sampling in mouse transplant models.</a> Leukemia &amp; lymphoma. 52, 1806-1808 (2011).</li><li>Warner, J. 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=Direct+evidence+for+cooperating+genetic+events+in+the+leukemic+transformation+of+normal+human+hematopoietic+cells.">Direct evidence for cooperating genetic events in the leukemic transformation of normal human hematopoietic cells.</a> Leukemia. 19, 1794-1805 (2005).</li><li>Chiu, P. P., Jiang, H., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia-initiating+cells+in+human+T-lymphoblastic+leukemia+exhibit+glucocorticoid+resistance.">Leukemia-initiating cells in human T-lymphoblastic leukemia exhibit glucocorticoid resistance.</a> Blood. 116, 5268-5279 (2010).</li><li>Guan, Y., Gerhard, B., Hogge, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+isolation,+and+stimulation+of+quiescent+primitive+leukemic+progenitor+cells+from+patients+with+acute+myeloid+leukemia+(AML).">Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML).</a> Blood. 101, 3142-3149 (2003).</li><li>Nolta, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gold+standard+improves:+a+better+assay+for+HSCs.">The gold standard improves: a better assay for HSCs.</a> Blood. 106, 1141-1142 (2005).</li><li>Kiel, M. J., 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=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li><li>Purton, L., Scadden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+factors+in+murine+hematopoietic+stem+cell+assays.">Limiting factors in murine hematopoietic stem cell assays.</a> Cell stem cell. 1, 263-270 (2007).</li><li>Dykstra, 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=Long-term+propagation+of+distinct+hematopoietic+differentiation+programs+in+vivo.">Long-term propagation of distinct hematopoietic differentiation programs in vivo.</a> Cell stem cell. 1, 218-229 (2007).</li><li>Jordan, C., Lemischka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+and+systemic+analysis+of+long-term+hematopoiesis+in+the+mouse.">Clonal and systemic analysis of long-term hematopoiesis in the mouse.</a> Genes &amp; development. 4, 220-232 (1990).</li><li>Challen, G., 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=Dnmt3a+is+essential+for+hematopoietic+stem+cell+differentiation.">Dnmt3a is essential for hematopoietic stem cell differentiation.</a> Nature genetics. 44, 23-31 (2012).</li><li>Hess, 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=Selection+based+on+CD133+and+high+aldehyde+dehydrogenase+activity+isolates+long-term+reconstituting+human+hematopoietic+stem+cells.">Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells.</a> Blood. 107, 2162-2169 (2006).</li><li>Mazurier, F., Doedens, M., Gan, O. I., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+myeloerythroid+repopulation+after+intrafemoral+transplantation+of+NOD-SCID+mice+reveals+a+new+class+of+human+stem+cells.">Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells.</a> Nat Med. 9, 959-963 (2003).</li></ol>

저자가 공개하는 게 없다.

직렬 BM 열망은 인간의 혈액 학적 장애의 임상 연구에 중요한 일상적인 절차입니다. 긴 실험을 통하여 세포의 구성 및 BM의 성분의 특성에 마우스에서 BM의 유사한 일련의 샘플링을 수행 할 수있는 능력 역시 매우 중요합니다. 이 절차는 마우스를 희생하지 않고 또한 말초 혈액의 내용이 BM에 거주하는 세포의 반사되지 않을 수 인스턴스에서 BM있는 추가적인 세포 유형의 존재를 검출하기위한 HSPC의 ...

All blood cells are derived from hematopoietic stem cells (HSCs). Procedures which allow access to the bone marrow (BM), the site of the vast majority of HSC&#8217;s in mice and men, without requiring sacrifice of animals, provide a resource to monitor the source of hematopoiesis serially. The overall goal of the procedure described here is to provide a detailed protocol for the sampling of BM cells from the femur of live mice which provides material representative of the cells which would be retrieved by conventional harvest of BM cells through sacrifice of the mice. The advantage of this procedure over conventional harvest of BM cells is that this procedure does not require the sacrifice of mice and therefore allows for longitudinal study of the bone marrow compartment of mice over time. 
Although this murine bone marrow aspiration procedure has been used in a number of studies and described previously (see Sundberg et al. for a historical review1), formal step-by-step procedures illustrating this technique have not been previously published. This protocol enables routine serial sampling of the BM for purposes such as assessing engraftment in the BM of cells exogenously introduced into the mouse (as would be done in xenograft studies for instance2,3), analysis of chimerism in the BM for comparison with that of the peripheral blood (where results are not necessarily congruent), monitoring the abundance of specific cell types normally enriched in the marrow such as hematopoietic stem and progenitor cells, and retrieval of BM cells for ex vivo culture and/or transplantation. In addition, evaluation of marrow contents may be very useful in murine models of hematological disease as aspiration is helpful in assessing hematological disease burden at time points when disease may not be evident in the peripheral blood3-5. Moreover, this technique may be used to evaluate response of hematologic disease in the marrow to drugs administered in therapeutic studies. Thus, the procedure described here is ideal for investigators wishing to obtain access to bone marrow hematopoietic cells from mice for cytological analysis, flow cytometric analysis, in vitro culturing, and/or in vivo transplantation studies without requiring sacrifice or harm to the mice. 
The protocol for sampling of BM described here is quite similar to the technique used for intra-femoral injection of material directly into the marrow cavity3,6. The key difference being that this protocol details the procedure for removal of cells from the marrow as opposed to instilling material directly into the marrow cavity space as is done with intrafemoral injection.

<table><tbody><tr><td>PBS</td><td>PAA</td><td>H15-002</td><td></td></tr><tr><td>Bovine serum albumin</td><td>PAA</td><td>K41-001</td><td></td></tr><tr><td>ACK lysis buffer</td><td>Homemade</td><td></td><td>in 1 L. Adjust pH 7.2 ~ 7.4 and filter sterile with 0.22 &amp;mu;m vacuum filter.</td></tr><tr><td>8.3 g Ammonium chloride</td><td>Fisher Scientific</td><td>A661-500</td><td></td></tr><tr><td>1 g Potassium bicarbonate</td><td>Fisher Scientific</td><td>P184-500</td><td></td></tr><tr><td>200 &amp;mu;l 0.5 M EDTA pH 8</td><td>Gibco</td><td>15575-038</td><td></td></tr><tr><td>RPMI 1640</td><td>PAA</td><td>E15-842</td><td></td></tr><tr><td>0.5 ml Tuberculin syringe 27.5 G</td><td>Becton Dickinson</td><td>305620</td><td></td></tr><tr><td>Sterile cell strainer 70 &amp;mu;m</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Attane</td><td>NDC:66794-014-25</td><td></td></tr><tr><td>Blunt-end needle</td><td>Stemcell Technologies</td><td>28110</td><td></td></tr><tr><td>PrecisionGlide needle 23 G</td><td>Becton Dickinson</td><td>305193</td><td></td></tr><tr><td>3 ml Syringe Luer-Lok tip</td><td>Becton Dickinson</td><td>309657</td><td></td></tr><tr><td>Non-tissue culture treated plate, 6 Well</td><td>Becton Dickinson</td><td>351146</td><td></td></tr><tr><td>12 x 75 mm 5 ml tubes&amp;nbsp;</td><td>Becton Dickinson</td><td>352054</td><td>FACS staining</td></tr><tr><td>12 x 75 mm 5 ml tubes with cell-strainer cap</td><td>Becton Dickinson</td><td>352235</td><td>FACS staining</td></tr><tr><td>NK1.1 APC-Cy7</td><td>Biolegend</td><td>108723</td><td></td></tr><tr><td>CD11b APC-Cy7</td><td>Biolegend</td><td>101225</td><td></td></tr><tr><td>CD45R (B220) APC-Cy7</td><td>Biolegend</td><td>103223</td><td></td></tr><tr><td>CD3 APC-Cy7</td><td>Biolegend</td><td>100222</td><td></td></tr><tr><td>Ly-6G and Ly-6C (Gr-1) APC-Cy7</td><td>Biolegend</td><td>108423</td><td></td></tr><tr><td>Ter119 APC-Cy7</td><td>Biolegend</td><td>116223</td><td></td></tr><tr><td>CD19 APC-Cy7</td><td>Biolegend</td><td>302217</td><td></td></tr><tr><td>CD4 APC-Cy7</td><td>BioLegend</td><td>317417</td><td></td></tr><tr><td>CD117 (c-KIT) PE</td><td>BioLegend</td><td>105808</td><td></td></tr><tr><td>Ly-6A/E (Sca-1) PE-Cy7</td><td>Biolegend</td><td>122513</td><td></td></tr><tr><td>CD34 APC</td><td>Biolegend</td><td>128612</td><td></td></tr><tr><td>CD16/32 e450</td><td>eBioscience</td><td>48-0161-82</td><td></td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma-Aldrich</td><td>32670</td><td></td></tr><tr><td>MethoCult GF M3434</td><td>STEMCELLTECHNOLOGIES</td><td>3434</td><td>For methocellulose culture</td></tr><tr><td>Carprofen</td><td>Crescent Chemical Company</td><td>C11045850</td><td>1 dose (5mg/kg)&amp;nbsp;</td></tr><tr><td>Flow cytometer, LSRFortessa</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Puralube vet ointment (sterile petrolatum ophthalmic ointment)</td><td>Dechra-US</td><td>17033-211-38</td><td></td></tr></tbody></table>

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.

라이브 C57/B6 마우스의 대퇴골 BM 흡입은 희생 후의 같은 종래의 마우스 BM 수확 하였다 BM 단핵 세포를 수득하는 데에 이용 하였다. 이 방법에 의해 얻어진 BM 단핵 세포이어서, BM 세포 (1) 세포 학적 분석에 의해 분석 (2) 조혈 줄기 / 전구 세포의 상대적인 주파수 (HSPCs)를 결정하고, 소트 HSPCs (3) 체외 배양 하였다. 후자의 실험, 혈통 부정적인 SCA1 + C-KIT에서 + (LSK) 세포는 BM 흡인에 의해뿐만 아니...

Watch this Scientific Journal Video about Femoral Bone Marrow Aspiration in Live Mice at JoVE.com

Femoral Bone Marrow Aspiration in Live Mice

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 ...

femoral-bone-marrow-aspiration-in-live-mice

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

37.8K 조회수.  Memorial Sloan-Kettering Cancer Center. 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.

비디오: Femoral Bone Marrow Aspiration in Live Mice

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

조혈 세포의 자동화 된 정량화 - Undecalcified 뼈의 조직 학적 이미지에서 기질 세포 상호 작용

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

연구

발생학

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Intravital 형광 현미경 검사 법 (IVFM)를 결합 하 여 유전자 모델 골 틈새에 조 혈 모 세포의 Engraftment 역학을 공부 하

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

뼈 전이의 모델

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

의학

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.

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 ...

면역학 및 감염병학

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

저자 스포트라이트: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies(쥐 혈액 악성 종양의 골수 미세환경 분석) 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

골수이형성 증후군 및 급성 골수성 백혈병의 골수 미세환경 집단 식별

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

암 연구학

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, ...

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 ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

라이브 쥐의 대퇴부 골수 흡인

요약

더 많은 비디오 탐색

조혈 세포의 자동화 된 정량화 - Undecalcified 뼈의 조직 학적 이미지에서 기질 세포 상호 작용

Intravital 형광 현미경 검사 법 (IVFM)를 결합 하 여 유전자 모델 골 틈새에 조 혈 모 세포의 Engraftment 역학을 공부 하

뼈 전이의 모델

마우스 골수 단핵구를 추적

골수이형성 증후군 및 급성 골수성 백혈병의 골수 미세환경 집단 식별

골수이형성 증후군 및 급성 골수성 백혈병의 골수 미세환경 집단 식별

골수이형성 증후군 및 급성 골수성 백혈병의 골수 미세환경 집단 식별

대한 대퇴골 창 회의소 모델

호중구 계보 세포의 질량 세포 분석 분석을위한 전체 골수의 준비

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis