نشكر التدفق الخلوي UMass الأساسية : ريتشارد Konz ، Giehl تيد غوسلين باربرا ، قو أوجدتها وKrupoch تامي. وقد تم تمويل هذا العمل من قبل المعاهد الوطنية للصحة / NHLBI RO1 HL084168 (MS) ، والمعاهد الوطنية للصحة CA - T32 130807 (JRS). كما استخدمت الموارد الأساسية التي تدعمها أبحاث الغدد الصماء السكري DK32520 منح المركز.

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	<li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+Fas-FasL+coexpression+by+erythropoietin+mediates+erythroblast+expansion+during+the+erythropoietic+stress+response+in.">Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in.</a> 108, 123-133 (2006).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Autoregulation+by+FAS+Mediates+Robust+Fetal+Erythropoiesis.">Negative Autoregulation by FAS Mediates Robust Fetal Erythropoiesis.</a> PLoS Biol. 5, e252-e252 (2007).</li><li>Krutzik, P. O., Hale, M. B., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+murine+immunological+signaling+network+with+phosphospecific+flow+cytometry.">Characterization of the murine immunological signaling network with phosphospecific flow cytometry.</a> J Immunol. 175, 2366-2373 (2005).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ineffective+erythropoiesis+in+Stat5a(-/-)5b(-/-)+mice+due+to+decreased+survival+of+early+erythroblasts.">Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.</a> Blood. 98, 3261-3273 (2001).</li><li>Guihard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAPK+ERK1+is+a+negative+regulator+of+the+adult+steady-state+splenic+erythropoiesis.">The MAPK ERK1 is a negative regulator of the adult steady-state splenic erythropoiesis.</a> Blood. 115, 3686-3694 (2010).</li><li>Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+erythroid+chaperone+that+facilitates+folding+of+alpha-globin+subunits+for+hemoglobin+synthesis.">An erythroid chaperone that facilitates folding of alpha-globin subunits for hemoglobin synthesis.</a> J Clin Invest. 117, 1856-1865 (2007).</li><li>Chen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythroid+dysplasia,+megaloblastic+anemia,+and+impaired+lymphopoiesis+arising+from+mitochondrial+dysfunction.">Erythroid dysplasia, megaloblastic anemia, and impaired lymphopoiesis arising from mitochondrial dysfunction.</a> Blood. 114, 4045-4053 (2009).</li><li>Chen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+distinct+stages+in+erythroid+differentiation+based+on+dynamic+changes+in+membrane+protein+expression+during+erythropoiesis.">Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.</a> Proc Natl Acad Sci U S A. 106, 17413-17418 (2009).</li><li>McGrath, K. E., Bushnell, T. P., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+hematopoietic+cells:+where+flow+meets+morphology.">Multispectral imaging of hematopoietic cells: where flow meets morphology.</a> J Immunol Methods. 336, 91-97 (2008).</li><li>Pop, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+key+commitment+step+in+erythropoiesis+is+synchronized+with+the+cell+cycle+clock+through+mutual+inhibition+between+PU.1+and+S-phase+progression.">A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression.</a> PLoS Biol. 8, (2010).</li><li>Borsook, H., Lingrel, J. B., Scaro, J. L., Millette, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+haemoglobin+in+relation+to+the+maturation+of+erythroid+cells.">Synthesis of haemoglobin in relation to the maturation of erythroid cells.</a> Nature. 196, 347-350 (1962).</li></ol>

وقد أجريت تجارب على الحيوانات وفقا للمبادئ التوجيهية واللوائح التي وضعتها جامعة ماساتشوستس اللجنة الطبية IACUC المدرسة.

منهجية تدفق cytometric التحقيق في وقت واحد يسمح لأي وظيفة الخلوية التي قد يتم الكشف عن الأجسام المضادة مع مضان ، مترافق محددة أو يجند ، بما في ذلك علامات سطح الخلية ، والتعبير من البروتين ، وبقاء الخلية ، مما يشير إلى الخلية باستخدام الفوسفات محددة الأجسام المضادة (3)...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> فهرس العدد </th></tr><tr><td> فاس ، البيوتين </td><td> دينار بحريني Pharmingen </td><td> 554256 </td></tr><tr><td> Streptavidin - APC </td><td> المسابر الجزيئية </td><td> S868 </td></tr><tr><td> 40 ميكرومتر مصفاة الخلية معقمة </td><td> Fisherbrand </td><td> 22363547 </td></tr><tr><td> البوليسترين جولة القاع أنابيب لتلطيخ FACS </td><td> فالكون دينار بحريني </td><td> 352008 </td></tr><tr><td> U - 96 لوحة أسفل جيدا </td><td> فالكون دينار بحريني </td><td> 353910 </td></tr><tr><td> ChromePure أرنب مفتش </td><td> جاكسون ImmunoResearch </td><td> 015-000-003 </td></tr><tr><td> CD71 - FITC (الأسهم 0.5mg/ml) </td><td> BD - العلوم البيولوجية </td><td> 553266 </td></tr><tr><td> Ter119 - PE (الأسهم 0.2mg/ml) </td><td> BD - العلوم البيولوجية </td><td> 553673 </td></tr><tr><td> 7AAD </td><td> BD - العلوم البيولوجية </td><td> 559925 </td></tr><tr><td> دابي مسحوق </td><td> روش </td><td> 236276 </td></tr><tr><td> FITC الجرذ المضادة للماوس CD41 MWReg30 </td><td> دينار بحريني Pharmingen </td><td> 553848 </td></tr><tr><td> FITC الجرذ المضادة للماوس CD45R/B220 RA3 - 6B2 </td><td> دينار بحريني Pharmingen </td><td> 553087 </td></tr><tr><td> FITC الجرذ المضادة للماوس CD411b/Mac-1 M1/70 </td><td> دينار بحريني Pharmingen </td><td> 557396 </td></tr><tr><td> FITC مكافحة الجرذ الفأر لي - 6G و Ly - 6C (GR - 1) RB6 - 8C5 </td><td> دينار بحريني Pharmingen </td><td> 553126 </td></tr><tr><td> FITC الهامستر المضادة للماوس CD3e 145 2C11 </td><td> دينار بحريني Pharmingen </td><td> 553061 </td></tr><tr><td> APC BrdU تدفق طقم </td><td> دينار بحريني Pharmingen </td><td> 557892 </td></tr><tr><td> Annexin V - البيوتين </td><td> دينار بحريني Pharmingen </td><td> 556418 </td></tr></tbody></table>

identification and analysis of mouse erythroid progenitors using the cd71/ter119 flow-cytometric assay

دراسة تكون الكريات الحمر يهدف إلى فهم الكيفية التي تتشكل الخلايا الحمراء من الأسلاف في وقت سابق للدم ومحمر. على وجه التحديد ، وينظم معدل تكوين الخلايا الحمراء التي إرثروبويتين هرمون (EPO) ، التي يتم تشغيلها بواسطة التوليف نقص الأكسجة الأنسجة. وثمة تهديد لائق نتائج الأوكسجين في الأنسجة من الزيادة السريعة في المكتب ، والقيادة زيادة في معدل الكريات الحمر ، وهي عملية تعرف باسم الاجهاد استجابة الكريات الحمر. الزيادة الناتجة في عدد خلايا الدم الحمراء المنتشرة يحسن نسيج تسليم الأوكسجين. فعال الاجهاد استجابة الكريات الحمر وبالتالي حاسمة بالنسبة لبقاء والتعافي من الظروف الفسيولوجية والمرضية مثل علو شاهق ، وفقر الدم ، والنزف ، والعلاج الكيميائي أو زرع الخلايا الجذعية. الفأر هو مفتاح نموذجا لدراسة تكون الكريات الحمر وردها الإجهاد. تكون الكريات الحمر الماوس النهائي (للبالغين من نوع) يأخذ مكان في كبد الجنين بين أيام الجنينية 12.5 و 15.5 ، في الطحال حديثي الولادة ، والكبار في الطحال ونخاع العظام. الطرق التقليدية لتحديد الأسلاف محمر في الأنسجة تعتمد على قدرة هذه الخلايا أن يؤدي إلى نشوء مستعمرات الخلايا الحمراء في وسائل الاعلام عندما مطلي شبه الصلبة المحتوية على المكتب. ويتم تحديد ذريتها السلائف محمر على أساس معايير شكلية. أيا من هذه الأساليب الكلاسيكية تسمح بالوصول إلى أعداد كبيرة من الخلايا محمر تمايز في مرحلة محددة للدراسة الجزيئية. نقدم هنا طريقة تدفق cytometric لتحديد ودراسة التمايز المرحلة الخاصة الأسلاف محمر والسلائف ، ومباشرة في سياق نسيج الماوس معزولة حديثا. في مقايسة يعتمد على علامات سطح الخلية CD71 ، Ter119 ، وعلى تدفق cytometric المعلمة &#39;إلى الأمام مبعثر&quot; ، والتي هي وظيفة من حجم الخلية. يمكن استخدام مقايسة CD71/Ter119 لدراسة الأسلاف محمر خلال استجابتها لإجهاد الكريات الحمر في الجسم الحي ، على سبيل المثال ، أو فقر الدم في الفئران الفئران الموجودة في ظروف انخفاض الأكسجين. ويمكن أيضا أن تستخدم لدراسة الأسلاف محمر مباشرة في أنسجة الفئران المعدلة وراثيا أو الأجنة ، بغية تقييم الدور المحدد للمسار الجزيئية تكون الكريات الحمر في تعديل.

Watch this Scientific Journal Video about Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay at JoVE.com

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

وهناك طريقة التدفق cytometric لتحديد وتحليل الجزيئي للتمايز في مرحلة معينة الأسلاف محمر الفئران والسلائف ، ومباشرة في طازجة تحصد الماوس نخاع العظم والطحال أو الكبد الجنين. في مقايسة يعتمد على سطح الخلية علامات CD71 ، Ter119 ، وحجم الخلية.

تحديد وتحليل الأسلاف محمر ماوس باستخدام CD71/TER119 التدفق cytometric الفحص

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

identification-analysis-mouse-erythroid-progenitors-using-cd71ter119

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Biology

31.8K Views.  University of Massachusetts Medical School. وهناك طريقة التدفق cytometric لتحديد وتحليل الجزيئي للتمايز في مرحلة معينة الأسلاف محمر الفئران والسلائف ، ومباشرة في طازجة تحصد الماوس نخاع العظم والطحال أو الكبد الجنين. في مقايسة يعتمد على سطح الخلية علامات CD71 ، Ter119 ، وحجم الخلية.

Video: Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

والفحص الكمية للأنسولين ، معربا عن الأسلاف لتشكيل مستعمرة

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

تدفق تحليل Cytometric من التكملة الإسفار ثنائي الجزيء: عالية الإنتاجية الأسلوب الكمي لدراسة التفاعل البروتين البروتين

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-Galactosidase القائم على فحص الفحص لتحديد الأدوية Senotherapeutic

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

تحليل سيتوميتريك من حويصلات خارج الخلية من خلية مكيفة وسائل الإعلام تدفق

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

تحديد وعزل وتوصيف السلف الليفية الشحمية (FAPs) والذرى العضلية المنشأ (أعضاء البرلمان) في العضلات الهيكلية في الجرذ

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

عزل السلف ميغاكاريوسيت الماوس

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...

التحليل الكمي للميتوفاجي في خلايا β البنكرياس الفئران

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

تسليط الضوء على المؤلف: تقييم تدفق الميتوفاجي في خلايا β البنكرياس باستخدام مناهج تكميلية فعالة وقوية 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

مناهج تكميلية لاستجواب تدفق الميتوفاجي في خلايا β البنكرياس

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

التحليل الكمي للتجميع الذاتي الميكروبي باستخدام قياس التدفق الخلوي للتصوير

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

تسليط الضوء على المؤلف: تطوير البحث في التجميع الذاتي الميكروبي باستخدام قياس التدفق الخلوي للتصوير

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

قياس التدفق الخلوي التصويري لدراسة التجميع الذاتي الميكروبي

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...