نشكر نوبويوكي Onai، تاكيزاوا هيتوشي ومانز ماركوس للحصول على المشورة الثمينة. وقد تم تمويل هذا العمل من قبل العلم السويسري مؤسسة وطنية، السويسري دوري السرطان وFondazione Ticinese في لوس انجليس من Ricerca Cancro سول.

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الإعلان عن أي تضارب في المصالح.

الطريقة الموصوفة هنا يمكن تحليل سريع ودقيق من تكون الدم في الفئران الفردية (الشكل 1). هذا التحليل في إعدادات التجريبية المختلفة، بما في ذلك نماذج الفئران من الالتهاب، والمناعة الذاتية، نقص المناعة، والأمراض التنكسية، واضطرابات الأيض والسرطان، ويتيح معالجة...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> RPMI 1640 </td><td> Gibco </td><td> 42401 </td></tr><tr><td> MEM NEAA 100X </td><td> Gibco </td><td> 11140 </td></tr><tr><td> الصوديوم البيروفات </td><td> Gibco </td><td> 11360 </td></tr><tr><td> PenStrep </td><td> Gibco </td><td> 15070 </td></tr><tr><td> برنامج تلفزيوني </td><td> Gibco </td><td> 20012 </td></tr><tr><td> FBS </td><td> Gibco </td><td> 16000 </td></tr><tr><td> خلية مصفاة 40 ميكرومتر </td><td> دينار بحريني الصقر </td><td> 352340 </td></tr><tr><td> 7-AAD تلطيخ حل </td><td> دينار بحريني Pharmingen </td><td> 559925 </td></tr><tr><td> ليز / فيكس العازلة </td><td> دينار بحريني Pharmingen </td><td> 558049 </td></tr><tr><td> بيرم العازلة الثالث </td><td> دينار بحريني Pharmingen </td><td> 558050 </td> &lt;/ TR&gt; <tr><td> كي-67 </td><td> دينار بحريني Pharmingen </td><td> 556026 </td></tr><tr><td> دابي </td><td> إينفيتروجن </td><td> D21490 </td></tr><tr><td> CD4 (GK1.5) </td><td> eBioscience </td><td> 150041 </td></tr><tr><td> CD8 (53-6،7) </td><td> eBioscience </td><td> 150081 </td></tr><tr><td> CD3 (145-2C11) </td><td> eBioscience </td><td> 150031 </td></tr><tr><td> CD45R (RA3-6B2) </td><td> eBioscience </td><td> 150452 </td></tr><tr><td> CD19 (6D5) </td><td> eBioscience </td><td> 150193 </td></tr><tr><td> GR1 (RB6-8C5) </td><td> eBioscience </td><td> 155931 </td></tr><tr><td> Tre119 (TER-119) </td><td> eBioscience </td><td> 155921 </td></tr><tr><td> NK-1.1 (PK136) </td><td> eBioscience </td><td> 455941 </td></tr><tr><td> ج كيت (2B8) </td><td> eBioscience </td><td> 171171 </td></tr><tr><td> هيئة السلع التموينية-1 (D7) </td><td> eBiosciسينعقد </td><td> 135981 </td></tr><tr><td> CD34 (RAM34) </td><td> eBioscience </td><td> 110341 </td></tr><tr><td> FcγR (2.4G2) </td><td> eBioscience </td><td> 553145 </td></tr><tr><td> مكافحة APC MicroBeads </td><td> Miltenyi بيوتيك </td><td> 130-090-855 </td></tr><tr><td> LS أعمدة </td><td> Miltenyi بيوتيك </td><td> 130-042-401 </td></tr></tbody></table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

نخاع العظام هو الموقع الرئيسي حيث HSCs وأكثر نضجا الأسلاف نسب خلايا الدم الموجودة وتفرق في كائن بالغ. HSCs يشكل سكان الخلية الدقيقة من الخلايا المحفزة قادرة على توليد خلايا الدم كل الأنساب لبعض الوقت والحياة 1. تشريح الجزيئي للتوازن HSCs في نخاع العظم لديها آثار مهمة في تكون الدم والأورام والطب التجديدي. وصفنا للبروتوكول وضع العلامات مع الأجسام المضادة فلوري والإجراءات المعزولة الإلكترونية في التدفق الخلوي ليسجل فرعية السلف المكونة للدم وتوزيع HSCs في الفئران الفردية (الشكل 1). وبالإضافة إلى ذلك، ونحن تصف طريقة لتخصيب على نطاق واسع الأسلاف المكونة للدم، وكذلك على المدى الطويل (LT) وعلى المدى القصير (ST) إعادة تشكيل HSCs من تجميع المعلقات العظام خلية نخاع بواسطة التخصيب المغناطيسي للخلايا التعبير عن C-كيت. يمكن استخدامها في إعداد الخلية الناتجة لفرز فرعية المختارة لفي المختبر، وفي الدراسات المجراة وظيفي (الشكل 2). كلا بانيات التربيقية 2،3 و 4 جيبية البطانة تشكل منافذ وظيفية دعم HSCs في نخاع العظام. عدة آليات في محراب بانية للعظم، بما في ذلك مجموعة فرعية من الخلايا بانية العظم + N-كادهيرين (3) والتفاعل بين مستقبلات التيروزين كيناز Tie2 أعرب في HSCs مع angiopoietin-1 يجند 5 منه نتفق في تحديد HSCs السكون. &quot;السبات&quot; في نخاع العظام أمر بالغ الأهمية لحماية HSCs من التكرار والإرهاق في نهاية المطاف على النشاط المفرط للدراجات 6. يمكن للمنبهات الخارجية تعمل على خلايا نظام المناعة الفطرية مثل يغاندس مستقبلات على الرقم (7) ومثل انترفيرون-α 6 تحفز أيضا الانتشار وتمايز HSCs إلى الأسلاف الملتزم النسب. مؤخرا، يبلغ عدد سكانها HSCs الماوس نائمة داخل لين - C-كيت + هيئة السلع التموينية-1 + CD150 + CD48 - CD34 - وصفت سكان 8. فرز الخلايا استنادا CD34 التعبير عن تعليق الخلية المكونة للدم الأسلاف التخصيب كما هو موضح هنا يسمح للعزلة هادئة على حد سواء الذاتي تجديد LT-HSCs وST-HSCs 9. وقد تم إجراء مماثل على أساس استنفاد الخلايا إيجابية نسب والفرز من LT-شهادة الثانوية العامة مع CD48 و Flk2 الأجسام المضادة التي سبق وصفها 10. في هذا التقرير نقدم بروتوكول للتوصيف المظهري والسابقين تحليل دورة الخلية الحية من أسلاف المكونة للدم، والتي يمكن أن تكون مفيدة لرصد تكون الدم في مختلف الظروف الفسيولوجية والمرضية. وعلاوة على ذلك، ونحن تصف FACS الفرز الداخلي لHSCs، والتي يمكن استخدامها لتحديد العوامل والآليات التي تنظم احترامهن لتجديد وتوسيع والتمايز في بيولوجيا الخلية والمقايسات نقل الإشارة، وكذلك للزرع.

Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

تم وصف طريقة لتحليل التوزيع من الأسلاف نخاع العظام المكونة للدم في التدفق الخلوي، وكذلك لعزل بكفاءة عالي النقاء الخلايا الجذعية المكونة للدم (HSCs). ويستند أساسا لإجراء العزل في تخصيب المغناطيسي للخلايا + C-كيت والخلية فرز لتنقية HSCs للدراسات الخلوية والجزيئية.

المظهري التحليل وعزل الخلايا الجذعية المكونة للدم الفئران والأسلاف النسب، الملتزم

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

phenotypic-analysis-isolation-murine-hematopoietic-stem-cells-lineage

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Biology

18.5K Views.  Bellinzona (Switzerland). تم وصف طريقة لتحليل التوزيع من الأسلاف نخاع العظام المكونة للدم في التدفق الخلوي، وكذلك لعزل بكفاءة عالي النقاء الخلايا الجذعية المكونة للدم (HSCs). ويستند أساسا لإجراء العزل في تخصيب المغناطيسي للخلايا + C-كيت والخلية فرز لتنقية HSCs للدراسات الخلوية والجزيئية.

Video: Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

العزلة وزرع الخلايا الجذعية المكونة للدم (HSCs)

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

اشتقاق خلايا الجذعية المكونة للدم من الفئران خلايا الجذعية الجنينية

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

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

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

العزلة وتحليل الخلايا الجذعية المكونة للدم من المشيمة

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

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

عزل الخلايا الجذعية المكونة للدم في وقت مبكر من الكيس المحي الفئران وAGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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

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

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

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

Lentiviral بوساطة خلال ضربة قاضية السابقين فيفو تكون الكريات الحمر في الإنسان خلايا الجذعية المكونة للدم

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

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

العزلة والثقافة، وزرع خلايا العضلات الأقمار الصناعية

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

عدوى فيروسات الفئران الجنينية الجذعية المستمدة خلية خلايا الجسم مضغي لتحليل التمايز لدم

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

تحديد العوامل الرئيسية تنظيم-تجديد الذات والتمايز في الخلايا المكونة للدم EML السلائف بواسطة تحليل الحمض النووي الريبي التسلسل

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

قياس الكتلة المتقدريه والاغشيه المحتملة في الخلايا الجذعية التي تكون الدم وخلايا T بواسطة التدفق الخلوي

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...