Name: femoral bone marrow aspiration in live mice
Uploaded: 2014-07-05T15:00:00.000+00:00
Duration: 7 min 10 s
Description: Watch this Scientific Journal Video about Femoral Bone Marrow Aspiration in Live Mice at JoVE.com

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.

وأجريت كافة الإجراءات الحيوانية وصفها في هذا البروتوكول وفقا للمبادئ التوجيهية لرعاية واستخدام الحيوانات المختبرية وتمت الموافقة من قبل لجنة رعاية الحيوان واستخدام المؤسسية (IACUCs كانت) في مركز ميموريال سلون كيترينج للسرطان. 1. تطلع نخاع العظم (BM) خلايا من عظم الفخذ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخدير الماوس الذي سيخضع الفخذ تطلع نخاع العظام مع isoflurane و(1-4٪) تدار مع المرذاذ الدقة. عمق التخدير وينبغي رصد كل 5-10 دقيقة في جميع أنحاء الداخلي من خلال مراقبة أنه لا يوجد تغيير في معدل التنفس المرتبطة التلاعب الجراحية و / أو الأذن، أخمص القدمين، وذيل قرصة. وبفعل التخدير مع isoflurane والمحافظة وأيضا في جميع أنحاء الإجراء مع isoflurane و. جرعة ما قبل إجرائية وقائية من البوبرينورفين بجرعة 0.05-0.1 ملغ / كغ تحت الجلد كل 6-12 ساعة يمكن استخدامها لمنع الألم الذي يصاحب الإجراء باعتباره صفةفريق الأمم المتحدة القطري إلى كاربروفين. </li><li style=";text-align:right;direction:rtl"> تطبيق مرهم للعين للعيون من الماوس الحث التالية التخدير لمنع جفاف القرنية. </li><li style=";text-align:right;direction:rtl"> يتم قص الفراء بعناية من مساحة الجلد ما يقرب من 150٪ أكبر من مساحة الموقع الطموح المنشود. تتم إزالة الفراء فضفاضة مع الشاش رطبة. يرجى الحفاظ على الماوس على لوحة تعميم الماء الدافئ أو غيرها من سطح يسيطر حراريا آمنة لمنع انخفاض حرارة الجسم أثناء العملية. </li><li style=";text-align:right;direction:rtl"> تطهير الساق بأكملها التي تحتوي على عظم الفخذ الذي سيخضع الطموح مع ثلاث مجموعات من الدعك بالتناوب (بالتناوب إما مع بوفيدون اليود (بتدين) أو الكلورهيكسيدين (Nolvasan) فرك و 70٪ ايزوبروبيل أو الايثانول 70٪ غارقة الإسفنج الشاش). </li><li style=";text-align:right;direction:rtl"> الرطب 0.5 مل حقنة السلين (حجم: 0.5 سم مكعب؛ قياس: 27.5 G) مع الفوسفات مخزنة المالحة عقيمة (PBS) قبل الشفط BM. ملء المحاقن مع 200-500 ميكرولتر من برنامج تلفزيوني وطرد على الفور برنامج تلفزيوني. كرر هذا الإجراء 2-3 روتتراوح. </li><li style=";text-align:right;direction:rtl"> إبقاء عازمة الساق من عظم الفخذ عن طريق دفع الساق مع أي البنصر أو الإصبع الخامس. تأكيد ان طائرة مخدر مناسبة تم تحقيقه. يقام الحقنة باستخدام الإبهام والسبابة. وهذا يسمح للاللقم أن تتعرض ويسهل إدخال الإبرة. </li><li style=";text-align:right;direction:rtl"> بعد ترطيب حقنة، أدخل الإبرة من خلال الوتر الرضفي بحيث يتم إيداع الإبرة بشكل آمن بين اثنين من اللقم من عظم الفخذ. من خلال عقد diaphysis بالقرب من الكردوس عظم الفخذ مع الإبهام والسبابة، يتم إدخال الإبرة إلى رمح من عظم الفخذ بسهولة. </li><li style=";text-align:right;direction:rtl"> قطب الإبرة إلى الخارج وإلى أعلى بحيث يكون بالتوازي مع رمح من عظم الفخذ. هذا الإجراء يسهل استرجاع محتويات نخاع العظام من عظم الفخذ رمح. </li><li style=";text-align:right;direction:rtl"> تحويل اتجاه عقارب الساعة وعكس عقارب الساعة إبرة بينما دفعت به ببطء في تجويف نخاع الفخذ. تأكيد الموضع الصحيح للإبرة عن طريق تحريك بلطف سيRINGE أفقيا. </li><li style=";text-align:right;direction:rtl"> برفق على المكبس إبرة الظهر، وخلق الضغط السلبي، في حين تتحرك الإبرة ذهابا وإيابا داخل تجويف BM. ملاحظة: إن حجم BM يستنشق تكون حوالي 5 ميكرولتر الذي يتوافق عادة إلى 0.4-0.8 × 10 6 خلايا وحيدات النوى. وسوف يتم تأكيد الطموح الناجح بصريا من خلال ظهور الدم في الجزء العلوي من الإبرة في قاعدة الحقنة. إذا رأيت أي الدم في حقنة فمن المرجح أن العظم أو الأنسجة جزء صغير عالق في الإبرة. هذا يمكن إزالته من الإبرة عن طريق تحريك المكبس صعودا وهبوطا في برنامج تلفزيوني (وهذا هو أحد الأسباب التي تجعل ينبغي حقنة مملوءة مسبقا مع برنامج تلفزيوني قبل الشفط BM). إذا لا يمكن فكها الأنسجة من حقنة، واستخدام إبرة حقنة جديدة و(مرة أخرى تبلل المحاقن مع 200-500 ميكرولتر من برنامج تلفزيوني). </li><li style=";text-align:right;direction:rtl"> مرة واحدة ويستنشق BM بنجاح من عظم الفخذ، وإزالة الإبر والمحاقن من عظم الفخذ والفأرة. </li><li style=";text-align:right;direction:rtl"> التحرك يستنشق BM إلى أنبوب microfuge العلاقات العامةefilled مع 500 ميكرولتر من برنامج تلفزيوني. بالنسبة لمعظم التطبيقات، ثم ينبغي أن تبقى الخلايا BM على الجليد حتى مزيد من المعالجة إذا كان ذلك ممكنا. </li><li style=";text-align:right;direction:rtl"> بعد الانتهاء من هذا الإجراء، وإدارة مسكن مع كاربروفين 5 ملغ / كغ تحت الجلد. ثم إزالة الماوس من التخدير ووضع على وسادة ساخنة حتى تعافى تماما. ملاحظة: يجب أن يكون هناك أي مضاعفات أو استغاثة من ذوي الخبرة باتباع الإجراء الطموح إذا فعلت بشكل صحيح. </li><li style=";text-align:right;direction:rtl"> قبل أن تعود الفئران إلى منطقة السكن، وضمان أنها قادرة على تجول والوصول إلى الطعام والماء. مراقبة الفئران لعلامات الضائقة أو إجراء آخر العدوى في ساعة 24 المقبلة. وتشمل علامات: النزيف المستمر، وفقر الدم والخمول. إذا رأيت أي من هذه العلامات إجراء آخر، يجب أن يتم التخلص من الحيوانات (ق). ملاحظة: يمكن تكرار تطلع BM / أخذ العينات ولكن يجب أن يتم تنفيذ الإجراء تكرار على عظم الفخذ المعاكس لمنع الصدمات المتكررة لنفس الساق. هناك القليل من المعلومات المتاحة عن الابequency أن تطلع BM لا يمكن أن يؤديها. ويتكرر الفخذ تطلع نخاع العظام عموما لا أكثر من مرة كل 2 أسابيع. </li></ol> 2. تقييم المحتوى الخلوي في خلايا يستنشق BM <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بيليه الخلايا التي تم استردادها من BM الطموح ومكان في أنبوب microfuge بواسطة الطرد المركزي في 300 x ج لمدة 5 دقائق في 4 درجة مئوية أو RT. </li><li style=";text-align:right;direction:rtl"> نضح طاف ثم resuspend وبيليه في 500 ميكرولتر من الدم الحمراء ACK العازلة تحلل الخلية (&quot;الأمونيوم كلوريد البوتاسيوم&quot; تحلل العازلة). </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا في الحمراء العازلة تحلل الخلية لمدة 10 دقيقة ثم قم بإضافة 1 مل من برنامج تلفزيوني وتدور باستمرار الخليط مرة أخرى في 300 x ج لمدة 5 دقائق في 4 درجة مئوية أو RT. ملاحظة: يتكون هي lysed بيليه خلايا الدم الحمراء من خلايا وحيدات النوى الآن BM. هذه يمكن معلق لFACS تلطيخ، عد الخلايا، زراعة الأعضاء، تحليل cytospin و / أو أي استخدام آخر (تماما كما خلايا BM تحصد من الفئران ضحى ستستخدم). </li></ol>

<ol>
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والكتاب ليس لديهم ما يكشف.

المسلسل الطموح BM هو إجراء روتيني حاسمة لتحقيق السريرية للاضطرابات الدموية في البشر. القدرة على إجراء أخذ العينات المسلسل شابهه من BM في الفئران لتوصيف التركيب الخلوي ومكونات BM طوال التجارب المطولة هو بالمثل قيمة للغاية. هذا الإجراء مفيد لتوصيف وHSPC دون التضحية الماوس...

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.

وقد استخدمت الفخذ BM تطلع ماوس C57/B6 الحية للحصول على خلايا وحيدات النوى BM تليها الحصاد BM التقليدية من نفس الماوس بعد التضحية. الخلايا وحيدة النواة BM حصلت عليها طريقتين ثم تم تحليلها من قبل (1) التحليل الخلوي للخلايا BM، (2) تحديد التكرار النسبي للخلايا الجذعية / السلف المك?...

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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.

الفخذ نخاع العظم الطموح في الفئران لايف

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

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Medicine

37.8K Views.  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.

Video: 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.

الكمي الآلي للخلية المكونة للدم - التفاعلات خلية انسجة في صور النسيجية من Undecalcified العظام

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

Developmental Biology

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.

الجمع بين الفحص المجهري نيون إينترافيتال (إيففم) مع النماذج الوراثية لدراسة ديناميات Engraftment الخلايا المكونة للدم لمنافذ نخاع العظام

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.

نماذج من الانبثاث العظام

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

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.

تتبع الماوس نخاع العظم وحيدات<em&gt; في فيفو</em

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

Immunology and Infection

عزل وتوصيف البيئة المكروية لنخاع العظم في الأورام النخاعية

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.

تسليط الضوء على المؤلف: تحليل البيئة المكروية لنخاع العظم في الأورام الخبيثة الدموية في الفئران 

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.

تحديد مجموعات البيئة الدقيقة لنخاع العظم في متلازمة خلل التنسج النقوي وسرطان الدم النخاعي الحاد

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

Cancer Research

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.

عظم الفخذ نافذة الغرفة نموذج ل<em&gt; في فيفو</em&gt; تتبع الخلايا في الفئران نخاع العظام

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.

إعداد نخاع العظام كله لتحليل الكتلة الخلايا من الخلايا العدلية النسب

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.

إجراءات زرع نخاع العظم في الفئران لدراسة الهيماتوبويز الكلونية

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

الفخذ نخاع العظم الطموح في الفئران لايف

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الكمي الآلي للخلية المكونة للدم - التفاعلات خلية انسجة في صور النسيجية من Undecalcified العظام

الجمع بين الفحص المجهري نيون إينترافيتال (إيففم) مع النماذج الوراثية لدراسة ديناميات Engraftment الخلايا المكونة للدم لمنافذ نخاع العظام

نماذج من الانبثاث العظام

تتبع الماوس نخاع العظم وحيدات

تحديد مجموعات البيئة الدقيقة لنخاع العظم في متلازمة خلل التنسج النقوي وسرطان الدم النخاعي الحاد

تحديد مجموعات البيئة الدقيقة لنخاع العظم في متلازمة خلل التنسج النقوي وسرطان الدم النخاعي الحاد

تحديد مجموعات البيئة الدقيقة لنخاع العظم في متلازمة خلل التنسج النقوي وسرطان الدم النخاعي الحاد

عظم الفخذ نافذة الغرفة نموذج ل

إعداد نخاع العظام كله لتحليل الكتلة الخلايا من الخلايا العدلية النسب

إجراءات زرع نخاع العظم في الفئران لدراسة الهيماتوبويز الكلونية