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<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 500 MLS של 0.22 פילטר מעוקרים ים מלאכותי מיקרומטר (FSW; מליחות 35 ppt). ים מלאכותי או טבעי סינון דרך מסנן 0.22 מיקרון מיקרומטר להסיר חלקיקים וחיידקים. </li><li style=";text-align:right;direction:rtl"> הרדימי אחד מבוגר הוואי bobtail דיונון (Euprymna scolopes) על ידי הצבת בפתרון 2% אתנול FSW. מניחים את החיה הרדמה למשך כ 10 דקות. דיונון תחדל שחייה לא יהיה פעיל להגיב למגע. הנשימה המשך, שמסומן על ידי התנועה של המעטפת, ופעילות chromatophore עדיין צריך להיות שנצפו. </li><li style=";text-align:right;direction:rtl"> המקום דיונון עם הצד הגחוני פונה כלפי מעלה על מגש שעווה רגילה לנתיחה. להטביע את חיה עם FSW המכיל אתנול 2%. </li><li style=";text-align:right;direction:rtl"> שימוש אחד רגיל 200 טיפים פיפטה μl, למשוך בחזרה את המשפך ואת המעטפת כדי לחשוף את כלי הדם הראשי cephalic ממוקם בין שתי העיניים. </li><li style=";text-align:right;direction:rtl"> באמצעות מזרק סטרילי 1 מ&quot;ל עם מחט 26.5-מד, לנקב את כלי הדם cephalic ולסגת בין 50-100 μl של hemolymph. מניחים את hemolymph בצינור סטרילי 1.5 מ&quot;ל על הקרח. הערה: אם חיה ישמש פעמים תורם רבות, רק לסגת 10-20 μl של hemolymph בכל זמן נתון. מחזירים את החיה טנק מי ים רגילים. חיה יהיה להחיות בתוך 30 דקות. </li><li style=";text-align:right;direction:rtl"> Hemocytes שנאספו טרי נשטפים מחדש תלויה μl 500 של פתרון קלמארי של Ringer (S-האצבעות; 530 mM NaCl, KCl 10 mM, 25 מ&quot;מ MgCl 2, 10 mM CaCl 2 ו - 10 mM חיץ HEPES, pH 7.5). </li><li style=";text-align:right;direction:rtl"> ריכוזים Hemocyte נקבעים על ידי hemocytometer, וכ 2,000 תאים מתווספים תלושי החדרים מכסה זכוכית, ואפשר לדבוק הזכוכית של 10 דקות בטמפרטורת החדר. בשלב זה צפיפות, hemocytes הטופס monolayer אחיד על פני השטח את השקופית זכוכית. </li><li style=";text-align:right;direction:rtl"> כדי לצפות חיידקי מחייב hemocytes המארח, hemocytes נחשפים זן חיידקי שכותרתו fluorescently כגון Vibrio fischeri ES114 ו / או Vibrio harveyi B392, שכל אחת מהן מכילה כתב פלואורסצנטי ירוק. V. fischeri ES114 וו &#39; harveyi B392 גדלים לשלב יומן אמצע ב tryptone מי ים מדיה (SWT) על 28 מעלות צלזיוס שייקר מסלולית. צפיפות אופטית ב 600nm נמדדת spectrophotometrically לקבוע צפיפות התאים. חיידקים הם pelleted ידי צנטריפוגה (5000 סל&quot;ד במשך 5 דקות), supernatant הוא מושלך, ואת גלולה מחדש תלויה S-האצבעות. </li><li style=";text-align:right;direction:rtl"> 100,000 תאים חיידקיים מתווספים קאמרית כל כך טוב כי יש 50 חיידקים לכל hemocyte בממוצע. Hemocyte / תערובות חיידקים מודגרת בפתרון של S-Ringer במהירות של 25 ° C עבור 1 שעה, זמן נקבע להניב את מרב מחייב. </li><li style=";text-align:right;direction:rtl"> הציטופלסמה של hemocytes אז הם מוכתמים fluorescently עם CellTracker אורנג&#39; 0.005% (Invitrogen) ולאחר מכן לשטוף S-האצבעות כדי להמחיש את התאים. </li><li style=";text-align:right;direction:rtl"> Hemocytes ויטראז עם חיידקים הקשורים נתפסים על ידי הקרינה או באמצעות גילוי Zeiss stereoscope V20 פלורסנט או מיקרוסקופ SP2 לייקה לייזר ספקטרלי confocal, ומנה מעל פני השטח של התא החי. </li></ol> נציג תוצאות מכיוון hemolymph תכשיטים hemocyanin מכיל תאית ולא המוגלובין, על חמצון, hemolymph יהפוך לכחול כהה. הממוצע של ~ 5000 hemocytes לכל μl של hemolymph יושג באמצעות שיטה זו. לאחר עמידה מחליק לכסות החדרים ואת מכתים ניאון, hemocytes אמור להופיע במאור אדום fluorescently ו amoeboid בצורת. עבור הידבקות חיידקים, V. fischeri תדבק גרוע hemocytes (1-2 תאים חיידקיים לכל תא דם) תוך V. harveyi תדבק מאוד (10-15 תאים חיידקיים לכל hemocyte). <img src="/files/ftp_upload/1714/1714fig1.jpg" alt="איור 1" /> באיור 1. למבוגרים הוואי bobtail דיונון Euprymna scolopes מראה המיקום של כלי דם cephalic. <img src="/files/ftp_upload/1714/1714fig2.jpg" alt="איור 2" /> איור 2. תוצאות של חשיפה hemocyte כדי Vibrio fischeri (א) ו Vibrio harveyi (B). אדום, כתום Cell Tracker, ירוק, GFP שכותרתו חיידקים.

<ol>
	<li>Koropatnick, T. A., Kimbell, J. R., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+host+hemocytes+during+the+initiation+of+the+squid-Vibrio+symbiosis.">Responses of host hemocytes during the initiation of the squid-Vibrio symbiosis.</a> Biol. Bull. 212, 29-39 (2007).</li><li>McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unseen+forces:+the+influence+of+bacteria+on+animal+development.">Unseen forces: the influence of bacteria on animal development.</a> Dev. Biol. 242, 1-14 (2002).</li><li>McFall-Ngai, M. J., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiont+recognition+and+subsequent+morphogenesis+as+early+events+in+an+animal-bacterial+mutualism.">Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism.</a> Science. 254, 1491-1494 (1991).</li><li>Nyholm, S. V., Stewart, J. J., Ruby, E. G., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+between+symbiotic+Vibrio+fischeri+and+the+haemocytes+of+Euprymna+scolopes.">Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes.</a> Environ. Microbiol. 11, 483-493 (2009).</li><li>Nyholm, S. V., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+winnowing:+establishing+the+squid-Vibrio+symbiosis.">The winnowing: establishing the squid-Vibrio symbiosis.</a> Nat. Rev. Microbiol. 2, 632-642 (2004).</li><li>Nyholm, S. V., Stabb, E. V., Ruby, E. G., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+animal-bacterial+association:+recruiting+symbiotic+Vibrios+from+the+environment.">Establishment of an animal-bacterial association: recruiting symbiotic Vibrios from the environment.</a> Proc. Natl. Acad. Sci. USA. 97, 10231-10235 (2000).</li><li>Nyholm, S. V., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+the+light-organ+microenvironment+of+Euprymna+scolopes:+description+of+a+population+of+host+cells+in+association+with+the+bacterial+symbiont+Vibrio+fischeri.">Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri.</a> Biol Bull. 195, 89-97 (1998).</li><li>Visick, K. L., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+fischeri+and+its+host:+it+takes+two+to+tango.">Vibrio fischeri and its host: it takes two to tango.</a> Curr. Opin. Microbiol. 9, 632-638 (2006).</li></ol>

מחקרים לגבי תפקידה של המערכת החיסונית בתיווך איתות מולקולרי בין חיידקים מועילים המארחים שלהם, בשנים האחרונות, תרומה משמעותית להבנת האבולוציה המשותפת של אאוקריוטים עם החיידקים שלהם. מערכת דיונון / Vibrio הוכיחה את עצמה כמערכת מודל ממושמע כדי לענות על שאלות היסוד בתחום זה 2,3,5,6,8. אור איבר של Euprymna דיונון scolopes היתרי קולוניזציה באופן בלעדי על ידי החיידק המאיר Vibrio fischeri. מאחר ורקמות שהבית חיידקים נשארים במגע עם מי ים, דיונון צריך לא רק לטפח את הסימביוזה ספציפיים, אלא גם להמשיך להוציא חיידקים אחרים. לימודי המשך חשפו כי macrophage כמו hemocytes סביר לשחק תפקיד חשוב בהקמת ותחזוקה של קשר זה 1,4,7. מכיוון מארח דיונון חסר חסינות אדפטיבית, הספציפיות מדהים נמצא קשר זה חייב להיות מלא או חלקי מתווכת דרך מערכת החיסון המולדת. החקירה האחרונה של תאים אלה בדם עולה כי hemocytes מבודד E. scolopes להכיר phagocytose V. fischeri ולא חיידקים סימביוטיים דיפרנציאלי והקולוניזציה כי סביר מוביל לסוג של &quot;סובלנות חיסונית&quot; של symbionts 4. פרוטוקול זה יהיה להדגים כיצד הצלחת להשיג את התאים האלה מהדם דיונון בוגר ולבדוק את יכולתם להיקשר חיידקים.

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<td>Lab-Tek chambered #1.0 borosilicate coverglass system (8-chambers)</td>
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<td>26.5 G 1ml latex-free insulin syringe</td>
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<td>Cell Tracker Orange</td>
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<td>Invitrogen</td>
<td>C34551</td>
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<td>SteREO Discovery V20 Microscope</td>
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<td>Zeiss</td>
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<td>SP2 Confocal Microscope</td>
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obtaining hemocytes from the hawaiian bobtail squid euprymna scolopes and observing their adherence to symbiotic and non-symbiotic bacteria

מחקרים לגבי תפקידה של המערכת החיסונית בתיווך איתות מולקולרי בין חיידקים מועילים המארחים שלהם, בשנים האחרונות, תרומה משמעותית להבנת האבולוציה המשותפת של אאוקריוטים עם החיידקים שלהם. הקשר הסימביוטי בין הוואי bobtail דיונון,<em&gt; Euprymna scolopes</em&gt; ואת החיידק bioluminescent<em&gt; Vibrio fischeri</em&gt; נוצל כמערכת מודל להבנת ההשפעות של חיידקים מועילים על התפתחות של בעלי חיים. מחקרים שנעשו לאחרונה הראו כי macrophage כמו hemocytes, המרכיב הסלולר הבלעדית של מערכת החיסון המולדת המארחת של דיונון, צפוי לשחק תפקיד חשוב בתיווך הקמת ותחזוקה של עמותה זו. פרוטוקול זה יהיה להדגים כיצד להשיג hemocytes מ<em&gt; א scolopes</em&gt; ולאחר מכן להשתמש תאים אלה מבחני מחייב חיידקי. למבוגרים דיונון מורדמים הראשון לפני hemolymph נאסף על ידי מזרק מתוך כלי הדם הראשי cephalic. Hemocytes המארח, הכלול hemolymph המחולץ, הם דבקו coverslips החדרים זכוכית חשוף ואז חלבון הפלואורסצנט הירוק שכותרתו הסימביוטי<em&gt; Vibrio fischeri</em&gt; ולא סימביוטית<em&gt; Vibrio harveyi</em&gt;. Hemocytes הם counterstained עם פלורסנט לצבוע (Cell Tracker אורנג&#39;, Invitrogen) ולאחר מכן דמיינו באמצעות מיקרוסקופ פלואורסצנטי.

Watch this Scientific Journal Video about Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria at JoVE.com

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

וידאו זה יהיה להדגים כיצד להשיג hemocytes (כדוריות דם) של הוואי bobtail דיונון,<em&gt; Euprymna scolopes</em&gt; לשימוש מבחני תא ביולוגי חיידקי הידבקות. Hemocytes יהיה מוכתם עם פלורסנט לצבוע וחשוף GFP שכותרתו חיידקים.

קבלת Hemocytes מן הוואי Bobtail קלמארי Euprymna scolopes והתבוננות דבקותם חיידקים סימביוטיים שאינן הסימביוטי

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

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Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

13.2K Views.  University of Connecticut. וידאו זה יהיה להדגים כיצד להשיג hemocytes (כדוריות דם) של הוואי bobtail דיונון, Euprymna scolopes לשימוש מבחני תא ביולוגי חיידקי הידבקות. Hemocytes יהיה מוכתם עם פלורסנט לצבוע וחשוף GFP שכותרתו חיידקים.

Video: Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

קבלת ביצים Xenopus laevis נקבות

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

ניסוחים להקפאת ייבוש של חיידקים והשפעתם על הישרדות תא

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

פרוטוקול פשוט לחילוץ Hemocytes מזחלי Wild

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

מדידת מוליכות עלה הידראוליים ומוליכות הפיוניות והתגובות שלהם לקרינה והתייבשות שימוש בשיטת אידוי השטף (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

בידוד ותרבות של Myofibers פרט ותאי הלווין שלהם משרירי שלד למבוגרים

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

אוסף, בידוד והעשרה של חיידקים המתרחשים באופן טבעי מMagnetotactic הסביבה

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

קבלת דגימות עם מואט, מואצת והפוך הזדקנות של דבורת דבש הדגם

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

שיטה לחילוץ פיגמנטים מן קלמארי<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

מערכת חלבון פלואורסצנטי ירוק לדמיין Effectors מועברת בין חיידקים במהלך זיהום

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

בידוד מיוציטים פרוזדוריים וחדריים באיכות גבוהה למדידות בו זמנית של Ca2+ Transients וזרם סידן מסוג L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...