ברצוננו להודות קתרינה Cosker וסטפני Courchesne לדיונים מועילים.

הכנה של ריאגנטים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ציפוי קולגן: קולגן מעיל P35 רקמה תרבות צלחות ומכניסים לתנור על 37 מעלות צלזיוס למשך 2 ימים לפני שמון חוצצים. הריכוז הסופי של קולגן צריך להיות 0.71 מ&quot;ג / מ&quot;ל ​​מדולל ב .001 N HCl. לאחר מכן, הוסף 1 מ&quot;ל של תערובת לכל צלחת. </li><li style=";text-align:right;direction:rtl"> מעמיסים גריז: כדי למלא את מטעין גריז, מזרק 60mL תחילה יש מלא שומן ואקום קורנינג. השתמש במזרק כדי למלא את מטעין גריז, לעטוף אותה בנייר כסף ואז החיטוי במשך 45 דקות. </li><li style=";text-align:right;direction:rtl"> טפלון חוצצים: חוצצים ניתן שימוש חוזר לאחר כל ניסוי, אך תחילה יש לנקות כראוי. הסר את המחיצה מהצלחת, לנגב את כל השומן הנותר ומניחים חומצה גופרתית עבור 2 ימים. לאחר הסרת מן החומצה, לשטוף עם מים לרתיחה 3X, במשך 20 דקות, לאפשר לו להתייבש, מקום צלחת פטרי P100 זכוכית החיטוי במשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> N2-methylcellulose: לשקול את 1.5g של methylcellulose ולמקם אותו בתוך בקבוק 500ml. הוספת בר מערבבים החיטוי אותו במשך 20 דקות על יבש (מנקודה זו כל העבודה חייב להיות סטרילי). לאחר מכן, להוסיף 500 MLS של נסיוב חינם התקשורת (N 2), ומערבבים בחדר קר עד שהוא נמס. Aliquot לתוך 50 מ&quot;ל conicals ולהקפיא ב -20 ° C. עבור aliquot עובד המניות, אחד conicals 50 מ&quot;ל לתוך צינורות 1mL ולהקפיא ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> DRG התקשורת: DMEM, 5% סוס מומת חום בדם, ו -1% סטרפטומיצין פניצילין. </li><li style=";text-align:right;direction:rtl"> 100ng/mL DRGN התקשורת: ריכוז המניות של שניהם גורם גדילה עצבי (NGF) ואת המוח נגזר גורם neurotrophic (BDNF) הוא 1mg/mL. מדולל לכל אחד neurotrophins 1:10,000 לתוך התקשורת DRG. תרבויות ניתן לגדל NGF לבד, זה משנה את המשלים של נוירונים לשרוד התרבויות. הערה: בעת הצורך, ריכוז (ציטוזין arabinoside) AraC הוא 1uM בשימוש בריכוז הסופי של 0.3uM. זה יהיה לעכב את הצמיחה של תאים שוואן ואת גליה אחרים. </li><li style=";text-align:right;direction:rtl"> 10ng/mL DRGN התקשורת: לדלל את DRGN 100ng/mL מדיה (01:10) עם התקשורת DRG. <img src="/files/ftp_upload/old/951_2008_10_27_6/951_Figure-01.png" alt="איור 1" width="392" height="294" /> באיור 1. כלים הדרושים הגדרת </li></ol> הגדרת לתאי מחולקים (להתחיל את התהליך הזה 1-2 ימים לפני הניתוח) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך לגרד באמצע צלחת מצופה קולגן P35 עם תנועה כלפי חוץ. </li><li style=";text-align:right;direction:rtl"> המקום 30ul של methylcellulose-N2 על באמצע השריטה. סט צלחות בצד עד המחיצה הוא משומן. </li><li style=";text-align:right;direction:rtl"> צרף מתאם 23-מד בדל luer כדי מטעין את המשחה. את מחיצת גריפ טפלון עם זוג 90 ° hemostats זווית ולהניח אותה שטוח עם מחיצת פונה כלפי מעלה תחת מיקרוסקופ. עקוב אחר המפריד עם גריז לוודא כי בכל פעם מתאם ממוקם בנקודת התחלה חדשה מתאם מוכנס לתוך שומן מן השלב הקודם, כך שיש קו רציף של שומן (ראה תרשים). לאחר גריז מוחל על המחיצה כולה, להפוך לאחד P35 מאכלים מוכנים הפוכים ולמקם אותו כך N2-methylcellulose נגמר בתא באמצע. לחץ כלפי מטה על תחתית צלחת עם פינצטה. הקפד ללחוץ על החלק הפנימי של המחיצה בארבע פינות (השמאלית העליונה, בפינה הימנית התחתונה, הימנית העליונה, השמאלית התחתונה, המצוין בתרשים על ידי &quot;X&quot;). <img src="/files/ftp_upload/old/951_2008_10_9_2/951_Figure-02.png" alt="איור 2" width="171" height="183" /> איור 2: צעדים כדי לשמן את המחיצה הערה: חשוב ללחוץ בחוזקה מספיק כך גריז עושה חותם להשלים עם המנה, אבל אם יותר מדי לחץ נוסף, האקסונים לא לחצות את הגבול לתוך התאים בצד. תרימי את hemostats, להפוך אותו שוב unclamp המפריד. לבסוף, במקום צלחת עם מחיצת מחובר היטב מתחת למיקרוסקופ התמקדות התחתון של התא האמצעי. עם מטעין את השומן, ליצור מחסום קטן (0.25 ס&quot;מ), כך שברגע התאים ממוקמים בתא באמצע שהם לא יכולים לדלוף החוצה. </li><li style=";text-align:right;direction:rtl"> לאחר להגדיר כמה תרבויות, DRG מקום התקשורת בכל התאים בצד ומקום באינקובטור שבו התאים יישמר. אפשר התרבויות לשבת במשך כמה שעות ואז לבדוק דליפה. אם בתקשורת יש דלף לתוך התא האמצעי, אז התרבות הוא שמיש. הערה: כשבני הראשון לומדים את הטכניקה הזו, חשוב להגדיר תרבויות יותר יש צורך בניסוי, כמו כמה יהיה דולף. <img src="/files/ftp_upload/old/951_2008_10_9_3/951_Figure-03.png" alt="איור 3" width="254" height="191" /> איור 3: &quot;טוב לעומת דולף&quot; תרבות </li></ol> נוירונים DRG שמירה בתרבויות מחולקים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יום 1: החלף DRG התקשורת התאים לצד 100ng/mL DRGN מדיה + AraC. בצע לנתיחה ולהוסיף תאים לתא מרכז (100,000 תאים). </li><li style=";text-align:right;direction:rtl"> יום 2: הוסף 10ng/mL מדיה + AraC החיצוני של המחיצה טפלון עד שהתקשורת זורמת מעל המכשול שומן ונוזלים חילופי עם תא מרכז. </li><li style=";text-align:right;direction:rtl"> יום 3: החלף התקשורת התאים צד 100ng/mL DRGN השמטת AraC ואת להקיף עם 10ng/mL DRGN השמטת AraC. </li><li style=";text-align:right;direction:rtl"> יום 6: החלף התקשורת התאים צד 1ng/mL + AraC ואת להקיף עם DRG מדיה + AraC. </li><li style=";text-align:right;direction:rtl"> יום 9: השתמש לניסויים. <img src="/files/ftp_upload/old/951_2008_10_9_5/951_Figure-04.png" alt="איור 4" width="267" height="148" /> איור 4: תמונות של גופות IHC התא אקסונים דיסטלי הערה: כאשר משנים את התקשורת, חשוב לשאוב את הנוזל מהחלק העליון של תא אחד בצד. כמו כן, לא לשנות את התקשורת מן התא האמצעי עצמו, רק להקיף את, ולתת לו לזרום מעל המכשול גריז למרכז. </li></ol>

<ol>
	<li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Control+of+the+Local+Environment+of+Somas+and+Neurites.">Independent Control of the Local Environment of Somas and Neurites.</a> Methods in Enzymology. 58, 302-307 (1979).</li><li>Watson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+use+the+Erk5+pathway+to+mediate+a+retrograde+survival+response.">Neurotrophins use the Erk5 pathway to mediate a retrograde survival response.</a> Nature Neuroscience. 4, 981-988 (2001).</li><li>Heerssen, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+motors+transport+activated+Trks+to+promote+survival+of+target-dependent+neurons.">Dynein motors transport activated Trks to promote survival of target-dependent neurons.</a> Nature Neuroscience. 7, 596-603 (2004).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2, 599-605 (2006).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 4, 2128-2136 (2006).</li><li>Ure, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+transport+and+steady-state+distribution+of+125I-nerve+growth+factor+in+rat+sympathetic+neurons+in+compartmented+cultures.">Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures.</a> J Neuroscience. 4, 1282-1290 (1997).</li></ol>

בסרטון הזה, אנחנו הוכיחו כיצד להכין ולתחזק לתאי מחולקים לשימוש נוירונים DRG culturing. בוצע כהלכה, מערכת זו מאפשרת הפרדה בין גוף התא של האקסון כדי ללמוד על המנגנונים שבאמצעותם neurotrophins אותות מעבר אקסונים ארוכים. מאז יש בידוד fluidic בין התאים, היא מאפשרת גירוי טיפול סלקטיבי או של תא אחד ללא...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>collagen</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>354249</td>
<td>&nbsp;</td>
<tr>
<td>N2-methylcellulose 400CPS</td>
<td>Reagent</td>
<td>Sel-Win Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Teflon divider</td>
<td>Other</td>
<td>Tyler Research</td>
<td>CAMP10</td>
<td>many other types of dividers are available</td>
</tr>
<tr>
<td>Pin rake</td>
<td>Tool</td>
<td>Tyler Research</td>
<td>Camp-PR</td>
<td>&nbsp;</td>
<tr>
<td>Grease loader</td>
<td>Tool</td>
<td>Tyler Research</td>
<td>Camp-GLSS</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Fisher</td>
<td>MT10017CV</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>450-01</td>
<td>&nbsp;</td>
<tr>
<td>BDNF</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>450-02</td>
<td>&nbsp;</td>
<tr>
<td>High vacuum grease</td>
<td>Reagent</td>
<td>Fisher</td>
<td>14-635-5D</td>
<td>&nbsp;</td>
<tr>
<td>AraC</td>
<td>Reagent</td>
<td>Sigma</td>
<td>C-1768</td>
<td>&nbsp;</td>
<tr>
<td>23 gauge luer stub adapter</td>
<td>Tool</td>
<td>Fisher</td>
<td>427565</td>
<td>&nbsp;</td>
<tr>
<td>90&deg; angle hemostats</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-7035</td>
<td>&nbsp;</td>		</tbody>
		</table>
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preparation and maintenance of dorsal root ganglia neurons in compartmented cultures

נוירונים להאריך תהליכים axonal כי הם רחוקים בגוף התא innervate רקמות היעד, שם היעד הנגזרות גורמי גדילה נדרשים להישרדות תפקוד עצבי ו. Neurotrophins נדרשים במיוחד כדי לשמור על בידול של הישרדות innervating עצב סנסורי אבל השאלה כיצד אלה יעד הנגזרות neurotrophins לתקשר אל גוף התא של הנוירונים innervating כבר על שטח של מחקר פעיל במשך 30 שנים. המודל המקובל בדרך כלל של כמה אותות neurotrophin להגיע לגוף התא מציעה כי איתות endosomes לשאת אות retrogradely לאורך האקסון. כדי ללמוד התחבורה מדרדר, מערכת התרבות פותחה במקור על ידי רוברט Campenot, שבו גופי התא מבודדים האקסונים שלהם. הטכניקה של הכנת אלה מחולקים לתאי עבור culturing נוירונים משחזר את הגירוי החושי סלקטיבית של מסופי נוירון המתרחשת בעקבות שחרור vivo של יעד הנגזרות neurotrophins. אירועים איתות מדרדר הדורשים ארוכי טווח תחבורה תלויה microtubule מדרדר להיות השלכות חשובות בטיפול בהפרעות ניוון עצבי.

Watch this Scientific Journal Video about Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures at JoVE.com

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

כאן אנו מתארים את הטכניקה של הכנת ושמירה לתאי מחולקים עבור נוירונים culturing חושית של הגרעינים השורש הגבי.

הכנה ותחזוקה של הגב הגרעינים נוירונים רוט בתרבויות מחולקים

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

preparation-maintenance-dorsal-root-ganglia-neurons-compartmented

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JoVE Journal

Biology

22.3K Views.  Dana Farber Cancer Institute. כאן אנו מתארים את הטכניקה של הכנת ושמירה לתאי מחולקים עבור נוירונים culturing חושית של הגרעינים השורש הגבי.

Video: Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

הכנת תרבויות העצבית מ Midgastrula עוברים שלב תסיסנית

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

הכנת ניתק תרבויות עכבר קליפתיים Neuron

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

הכנת כדורי אקדח ין transfection Biolistic של נוירונים תרבות Slice

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

הכנת 2-dGuo שטופלו תרבויות איברים קורנית

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

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 ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

הכנת Aplysia חושי, מנוע תא תרבויות העצבית

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

תרבויות אלטרנטיבי לפלוריפוטנטיים אנושיים לתאים גזע ייצור, תחזוקה וניתוח גנטי

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

מערכת עין מתנהל שורש לרוחב ב<em&gt; ארבידופסיס</em&gt; ותירס

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

סטייה של מיקרוביוטה שורש בבתי גידול שונים המבוססת על רשתות מתאם משוקללים

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

בדיקת כימוטקסיס משופרת לזיהוי מהיר של כימותרפים ריזובקטריאליים באקסודות שורש

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...