الكتاب أود أن أشكر الدكتور شينج دينج من معهد سكريبس للبحوث والدكتور دنغ Hongkui من جامعة بكين للحصول على المساعدة والتوجيه.

1. إعداد لوحات التغذية MEF <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل يوم واحد الخلايا ES زراعة ، أذاب one قنينة من E14.5 المشع CF - 1 الخلايا المغذية MEF (انظر إجراءاتنا بشأن كيفية أذاب خلايا ES). </li><li style=";text-align:right;direction:rtl"> لوحة MEF المغذية للخلايا في مناطق ذات كثافة من 10000 خلية / كذلك في لوحة 12 أيضا. احتضان الخلايا ليلا 37 درجة مئوية و 5 ٪ CO 2. MEF الخلية وسائل الإعلام : DMEM تستكمل مع 10 ٪ ES - برنامج تلفزيوني ، والجلوتامين 1X 1X غير الأحماض الأمينية الأساسية. </li></ol> 2. الحفاظ على التجديد الذاتي للخلايا ES ماوس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فصل الخلايا ES باستخدام التربسين. بذور الخلايا في مناطق ذات كثافة من 50000 خلية / كذلك على لوحات معدة مسبقا MEF تغذية النمو في وسائل الإعلام (MEM خروج المغلوب تستكمل مع KSR 15 ٪ ، 4 مم الجلوتامين L - ، 0.1 ملم غير الاساسيين من الأحماض الأمينية وBME 1X) تستكمل مع SC1 على التركيز المطلوب (100 نانومتر كنا ، NM 300 ، وميكرومتر 1). علينا أيضا أن أضيف مراقبة إيجابية جيدا أن استكمل مع 1000 μ / مل من LIF. احتضان الخلايا ليلا 37 درجة مئوية و 5 ٪ CO 2. </li><li style=";text-align:right;direction:rtl"> ثقافة الخلايا عند 37 درجة مئوية و 5 ٪ CO 2 لمدة 3-4 أيام في كل عبور. تحديث وسائل الإعلام كل 48 ساعة. </li><li style=";text-align:right;direction:rtl"> خلايا مرور 1:10 حتي 1:15 التربسين مع الحفاظ على كثافة مماثلة لكثافة الغرس الأولي. بعد 5 فقرات ، يمكن فحص الخلايا للتعبير عن علامات تعدد القدرات باستخدام كيمياء سيتولوجية مناعية نموذجية. </li></ol> 3. Immunocytochemical فحص علامات تعدد القدرات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسل الخلايا برفق ثلاث مرات مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> تحديد الخلايا مع 500 ميكرولتر من مثبت لمدة 20 دقيقة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> غسل الخلايا برفق ثلاث مرات مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> كتلة غير محددة وملزمة مع العازلة ميكرولتر 500 حظر لمدة ساعة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا مع 250 ميكرولتر من الأجسام المضادة الأولية محددة (كنا Nanog ، Sox2 ، SSEA1 وOct4 في التخفيفات التالية : 1:500 ، 1:2000 ، 1:500 ، و1:500) </li><li style=";text-align:right;direction:rtl"> غسل الخلايا برفق ثلاث مرات مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا مع 250 ميكرولتر من الأجسام المضادة الثانوية لمدة ساعتين في درجة حرارة الغرفة ، والابتعاد عن الضوء (استخدمنا الحمير لمكافحة فأر مترافق مع فلور اليكسا ® 555 في 1:2000 والحمار المضادة للأرنب مترافق مع فلور اليكسا ® 488 في 1 : 2000). </li><li style=";text-align:right;direction:rtl"> غسل الخلايا برفق ثلاث مرات مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> إضافة دابي (تركيز النهائي 1 ميكروغرام / مل) لغسل الماضي ، واحتضان 5 دقائق لتصور النوى. </li><li style=";text-align:right;direction:rtl"> تحليل الخلايا تحت المجهر الفلورسنت. </li></ol> 4. ممثل النتائج : 1. مورفولوجيا النتائج : ونمت الخلايا الماوس ES (ESC R1) على الخلايا المغذية MEF في وجود إما LIF SC1 أو للحفاظ على الذات في تجديد حالة غير متمايزة. بعد مرور الثانية ، يمكن ملاحظة التمايز في الخلايا دون LIF أو SC1 (السيطرة السلبية) ، وبعد 5 الممرات وقد لوحظت في الأساس ليست مستعمرات غير متمايزة. بقي LIF (مراقبة إيجابية) مستعمرات تعامل في حالة غير متمايزة في جميع أنحاء الممرات الخلية 5. خلايا تعامل مع SC1 بتركيزات من 300 نيوتن متر أو 100 نانومتر كما بقيت غير متمايزة بعد 5 الممرات ، ومع ذلك ، فإن الخلايا المعالجة مع 1 ميكرومتر SC1 شهدت موت الخلية بعد مرور الرابع. (أنظر الشكل 2 من مذكرة التطبيق) ويلخص الجدول 1 الملاحظات الشكلية. 2. التعبير عن علامات تعدد القدرات تم إصلاح خلايا الفأر ES المحافظة لمدة 5 الممرات وفحصها للكيمياء سيتولوجية مناعية SSEA1 ، Nanog ، وOct4 Sox2. وكان التعبير علامة مشابهة للخلايا في الحفاظ LIF. (أنظر الشكل 3 و 4 من مذكرة التطبيق) <img src="/files/ftp_upload/1550/1550fig2a.jpg" alt="الشكل 1" /> الشكل 1 : مقارنة مورفولوجيا الخلايا أو الاحتفاظ بها في LIF SC1. لم يلاحظ أي اختلاف في التشكل. <img src="/files/ftp_upload/1550/1550fig2c.jpg" alt="الشكل 2 و 3" /> الشكل 2 والشكل 3 : Nanog ، SSEA1 ، Sox2 ، وتلطيخ Oct4 الخلايا الحفاظ على وفاق مع SC1 أو LIF (التين 4 من مذكرة التطبيق). في الحفاظ على خلايا مماثلة التعبير SC1 تظهر علامة على الخلايا الحفاظ على تعدد القدرات في LIF. الجدول 1 <table border="1" cellspacing="0" cellpadding="0" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> التحكم السلبي </td><td> LIF التحكم ايجابي </td><td> SC1 1uM </td><td> SC1 300 نانومتر </td><td> SC1 100 نانومتر </td></tr><tr><td> 1 الممر </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td></tr><tr><td> 2 الممر </td><td> بعض مورفولوجيا متباينة </td><td> البريد العاديS خلية مورفولوجيا </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td></tr><tr><td> 3 ممر </td><td> الغالبية العظمى من الخلايا المتمايزة </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td></tr><tr><td> 4 ممر </td><td> لا مستعمرات ESC </td><td> مورفولوجيا الخلايا العادية ES </td><td> لوحظ موت الخلية </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td></tr><tr><td> 5 الممر </td><td> متمايزة </td><td> مورفولوجيا الخلايا العادية ES </td><td> لوحظ موت الخلية </td><td> مورفولوجيا الخلايا العادية ES </td><td> مورفولوجيا الخلايا العادية ES </td></tr></tbody></table>

<ol>
	<li>Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A., Gough, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336, 684-687 (1988).</li><li>Ying, Q. L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., Smith, 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+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453, 519-523 (2008).</li><li>Chen, S., Do, J. T., Zhang, Q., Yao, S., Yan, F., Peters, E. C., Scholer, H. R., Schultz, P. G., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+embryonic+stem+cells+by+a+small+molecule.">Self-renewal of embryonic stem cells by a small molecule.</a> Proc. Natl. Acat. Sci. USA. 103, 17266-17271 (2006).</li></ol>

ويعمل واضعو هذه المادة التي تنتج Stemgent الكواشف والأدوات المستخدمة في هذه المقالة.

يمكن استخدام SC1 جزيء صغير للحفاظ على الذات التجديد مع دولة غير متمايزة في الخلايا ES الماوس. قبل استخدام على خط الخلية التي لم تختبر ، ومع ذلك ، ينبغي معاير لتحديد تركيز الأمثل. على سبيل المثال ، نحن اختبار ثلاثة تركيزات على الخلية ES الماوس R1 ESC الخط. 100 نانومتر و 300 نانومتر التركيزات كانت قادرة على الحفاظ على خلايا ES الماوس ، ومع ذلك ، كان التركيز فيها 1 ميكرومتر السامة. ويمكن أيضا أن تستخدم تحت SC1 تغذية خالية من الشروط.

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<td>Santa Cruz</td>
<td>21702</td>
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<td>Nanog antibody</td>
<td>&nbsp;</td>
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<td>ab21603</td>
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the use of sc1 (pluripotin) to support mesc self-renewal in the absence of lif

تستزرع تقليديا الماوس الجذعية الجنينية (ES) مع خلايا اللوكيميا عامل المثبط (LIF) للحفاظ على الذات التجديد.<sup&gt; 1</sup&gt; ومع ذلك ، LIF مكلفة وليس تفعيل المسار LIF/JAK/STAT3 المطلوب تماما للحفاظ على حالة التجديد الذاتي.<sup&gt; 2</sup&gt; قد يكون جزيء صغير SC1 بديلا اقتصاديا لLIF. وظائف عن طريق تثبيط SC1 المزدوج للرأس وجاب ERK1.<sup&gt; 3</sup&gt; رسم توضيحي لآلية عملها يجعلها أداة مفيدة لدراسة الآلية الجزيئية الأساسية التجديد الذاتي. نحن هنا لشرح الداخلي للخلايا الفأر في زراعة وفاق وجود SC1 وتظهر أنها قادرة على الحفاظ على تجديد الذاتي في غياب LIF. وأظهرت الخلايا المستزرعة مع SC1 التشكل مماثلة مقارنة مع المحافظة على الخلايا LIF. عرضت كلا نموذجي مورفولوجيا ES الماوس بعد خمس فقرات. ولوحظ تعدد القدرات التعبير عن علامات نموذجي (Oct4 ، Sox2 ، Nanog وSSEA1) بعد خمس فقرات في حضور SC1. وعلاوة على ذلك ، تسبب أي سمية SC1 العلني على خلايا ES الماوس.

Watch this Scientific Journal Video about The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF at JoVE.com

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

وظائف عن طريق تثبيط SC1 المزدوج للرأس وجاب ERK1. نحن اختبار وظيفة SC1 في دعم خلية فأر ES التجديد الذاتي في غياب LIF وأظهرت أن SC1 غير قادرة على الحفاظ على الذات تجديد الخلايا ES الثقافات الماوس.

استخدام SC1 (Pluripotin) لدعم MESC التجديد الذاتي في غياب LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

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The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

10.5K Views.  Stemgent. وظائف عن طريق تثبيط SC1 المزدوج للرأس وجاب ERK1. نحن اختبار وظيفة SC1 في دعم خلية فأر ES التجديد الذاتي في غياب LIF وأظهرت أن SC1 غير قادرة على الحفاظ على الذات تجديد الخلايا ES الثقافات الماوس.

Video: The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

استخدام أدوات الكشف البروتياز نيون تحديد نشاط الأنزيم البروتيني

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

استخدام بيوفيدباك في الواقع الافتراضي السريرية : مشروع مقدام

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

الكمي بؤر γH2AX في الاستجابة للإشعاع المؤين

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Oct4GiP الفحص مراسل لدراسة الجينات التي تنظم الفأر الخلايا الجذعية الجنينية الصيانة والتجديد الذاتي

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

استخدام الكيموستات في الميكروبية بيولوجيا الأنظمة

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

استخدام الأنزيمية أجهزة الاستشعار لقياس الذاتية ATP أو H<sub&gt; 2</sub&gt; O<sub&gt; 2</sub&gt; في الكلى

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

تطوير نظام إدراج المشارك ثقافة نوعين الخلوية في غياب خلية خلية الاتصال

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

استخدام<em&gt; فيفو السابقين</em&gt; التصوير الجامع بالأعضاء والأنسجة الكمي الأنسجة لتحديد بيو توزيع الجزيئات Fluorescently إعتبر

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...