<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קח spleens לואיס חולדה (חולדות בין 160-200 הם הטובים ביותר). Dilacerate על קרח בצלחת פטרי המכילות PBS + אנטיביוטיקה (PBS-PS) בעזרת מסננת התא. שים בתוך שפופרת 50 מ&quot;ל. מלא עם PBS-PS. </li><li style=";text-align:right;direction:rtl"> ספין של 10 דקות ב 4 ° C עד גלולה התאים. </li><li style=";text-align:right;direction:rtl"> לשטוף את התאים פעמיים. </li><li style=";text-align:right;direction:rtl"> Resuspend גלולה ב NH 4 Cl 0.15 מ &#39;(5 מ&quot;ל לכל טחול). מערבבים בעדינות עם pipet ברציפות למשך 3 דק &#39;על קרח lyse אריתרוציטים. מלאו את הצינור עם המדיום. </li><li style=";text-align:right;direction:rtl"> ספין של 10 דקות ב 4 ° C עד גלולה התאים. </li><li style=";text-align:right;direction:rtl"> לשטוף את התאים פעמיים. </li><li style=";text-align:right;direction:rtl"> ספירת התאים. הטחול עכברוש נותן 200-250000000 תאים. </li><li style=";text-align:right;direction:rtl"> זרעים התאים ב -2 מיליון דולר מ&quot;ל במדיום שלם: 50 מ&quot;ל לכל בקבוק 75 ס&quot;מ 2. </li><li style=";text-align:right;direction:rtl"> תנו לגדול במשך 48 שעות בחממה. </li><li style=";text-align:right;direction:rtl"> ספין 15 דקות ב 4 ° C עד גלולה התאים. </li><li style=";text-align:right;direction:rtl"> אסוף את supernatant; להשליך את התאים. </li><li style=";text-align:right;direction:rtl"> הוסף 15 מ&quot;ג / מ&quot;ל ​​mannoside מתיל אל supernatant. מערבבים היטב. </li><li style=";text-align:right;direction:rtl"> מסנן (0.2 מ&quot;מ) </li><li style=";text-align:right;direction:rtl"> Aliquot ולאחסן ב -20 ° C. יכול להיות כל הזמן על 4 מעלות צלזיוס למשך 10 ימים במקרה הצורך. </li></ol>

<ol>
	<li>Beeton, C., Barbaria, J., Devaux, J., Benoliel, A. -. M., Gola, M., Sabatier, J. -. M., Bernard, D., Crest, M., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blocking+of+voltage-gated+K++channels+treats+experimental+autoimmune+encephalomyelitis+and+inhibits+T-cell+activation.">Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation.</a> J. Immunol. 166, 936-944 (2001).</li><li>Beeton, C., Wulff, H., Barbaria, J., Clot-Faybesse, O., Pennington, M., Bernard, D., Cahalan, M. D., Chandy, K. G., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blockade+of+T+lymphocyte+K++channels+ameliorates+experimental+autoimmune+encephalomyelitis,+a+model+for+multiple+sclerosis.">Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis.</a> Proc. Natl. Acad. 98, 13942-13947 (2001).</li><li>Mor, F., Cohen, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+Lewis+rat+encephalitogenic+T+cell+lines:+T-cell-growth-factor+is+superior+to+recombinant+IL-2.">Propagation of Lewis rat encephalitogenic T cell lines: T-cell-growth-factor is superior to recombinant IL-2.</a> J. Neuroimmunol. 123, 76-82 (2002).</li></ol>

The authors have nothing to disclose.

אנחנו מכינים TCGF מ splenocytes לואיס עכברוש מאז שאנחנו בקביעות להרדים חולדות נאיבי מהמאמץ הזה לקצור בסרום thymi לעורר לואיס עכברוש T שורות תאים במבחנה. TCGF זה יכול לשמש כדי לקדם את הצמיחה ואת ההישרדות של שורות תאים T מ זני עכברים אחרים. TCGF יכול גם להיות מוכנים מן זנים אחרים של חולדות.

כדי להכין מדיום תרבות להשלים את הפעולות הבאות כדי להוסיף בקבוק 500 מ&quot;ל של RPMI 1640 וסטרילי, מסנן: FCS 10%; 1 בקבוק סן ז&#39;רמן: 5 מ&quot;ל ויטמינים RPMI; 5 נתרן pyruvate מ&quot;ל, 5 מ&quot;ל שאינם חיוניים חומצות אמינו, 50 אממ בטא mercaptoethanol; 2 UG / ml Concanavalin א

preparing t cell growth factor from rat splenocytes

תחזוקה של אנטיגן ספציפי לערמונית T שורות תאים או שיבוטים בתרבות דורש הפעלת סיבובים של אנטיגן-Induced מופרדות שלבי 1,2 הרחבת התא. תוספת של אינטרלוקין 2 לתקשורת התרבות בשלב ההתרחבות נחוצה כדי למנוע מוות של תאים ו מספיק כדי לשמור לטווח קצר שורות תאים T אבל הוכח להגדיל Th1 קיטוב 3. החלפה של אינטרלוקין 2 בפקטור תא T צמיחה (TCGF) אשר מכיל תערובת של ציטוקינים הוא יעיל יותר מאשר אינטרלוקין 2 בשמירה לטווח ארוך שורות תאים T במבחנה 3. יתר על כן, TCGF יכול בקלות להיות מוכנים בכמויות גדולות במעבדה, והוא הרבה יותר זול מאשר רקומביננטי אינטרלוקין 2. כאן, אנו מראים כיצד להכין TCGF מ supernatants עכברוש splenocyte תרבות. במשך התהליך הזה, שאנחנו קוצרים spleens מ נאיבי חולדות לואיס מורדמים עבור התימוס ואיסוף הדם. אנחנו מכינים תא בודד השעיות מן spleens, lyze את כדוריות הדם האדומות של הלם אוסמוטי, ואת הזרע splenocytes במדיום תרבות. התאים מגורה עם concanavalin, mitogen כי הלא סלקטיבי מפעיל כל T לימפוציטים עכברוש, וישכנע את הייצור של ציטוקינים. Supernantant תרבות נאסף 48 שעות לאחר מכן concanavalin andexcess חייב mannoside מתיל אלפא כדי למנוע את הפעלת הקווים תא T אשר TCGF יתווספו. TCGF אז הוא סטרילי, מסונן, aliquoted, ומאוחסנים ב -20 ° C.

Watch this Scientific Journal Video about Preparing T Cell Growth Factor from Rat Splenocytes at JoVE.com

Preparing T Cell Growth Factor from Rat Splenocytes

אנו מתארים את הכנת גורם תא T צמיחה המשמש הרחבה של מבחנה של אנטיגן ספציפי לערמונית קווי עכברוש T לימפוציטים.

הכנת פקטור T צמיחת תאים מן Splenocytes עכברוש

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

preparing-t-cell-growth-factor-from-rat-splenocytes

Nanyang Technological University

Research

JoVE Journal

Biology

12.6K Views.  University of California, Irvine (UCI). אנו מתארים את הכנת גורם תא T צמיחה המשמש הרחבה של מבחנה של אנטיגן ספציפי לערמונית קווי עכברוש T לימפוציטים.

Video: Preparing T Cell Growth Factor from Rat Splenocytes

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

פקטור מצופה צמיחה השמה ביד על Explants forebrain הגבי

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

הכנת E18 נוירונים עכברוש קליפתיים עבור מידור בתוך המכשיר microfluidic

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

הצבת פקטור מצופה חרוזים צמיחה על עוברי תרנגולת בשלב מוקדם

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

תא תרבויות העצבית מ Aplysia עבור הדמיה ברזולוציה גבוהה של קונוסים צמיחה

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

פרוטוקול נוירונים culturing הסימפתטית מן הגרעינים עכברוש צוואר הרחם מעולה (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

הדמיה חיה של שחול Cell מן האפידרמיס של דג הזברה פיתוח

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

השתנה שמרים-Two-Hybrid System כדי לזהות חלבונים אינטראקציה עם גורם הגדילה Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

הכנת הפרט תסיסנית ביצה צ&#39;יימברס הדמיה חיים

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

ניתוח קינמטית של חטיבה ואת הרחבת תא: כימות הבסיס הנייד של אזורי התפתחותית צמיחת הדגימה<em&gt; Zea Mays</em&gt; מותיר

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

אנליזה מולקולרית של אנדותל-mesenchymal מעבר המושרה על ידי הפיכת גורם גידול-β איתות

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...