הופק על ידי Loredana Manfra של מכון המחקר להגנה על הסביבה (Ispra-מחלקת ניטור איכות הסביבה) ו פדריקה Savorelli של הסוכנות האזורית לאיכות הסביבה (Emilia-Romagna, איטליה). התייחסות חברי עבור קבוצת המשנה העבודה על הארטמיה לסרטן בקבוצת שיטות ביולוגיות - מים מלוחים / מים מליחים ומשקעים של ועדת UNICHIM על איכות המים (המכון הפעלה תקינה טכנית במגזר כימי). Videography ועריכה על ידי מרקו Pisapia של Ispra (יחידת האינטרנט). תיאום הפקה של אנה מריה קיקרו אריקה Magaletti של Ispra (מחלקת ניטור איכות הסביבה). אנו רוצים להודות הסוכנות האזורית לאיכות הסביבה של אמיליה, רומניה, פררה סניף, קבוצת שיטות ביולוגיות - מים מלוחים / מליחיםמים ומשקעים של ועדת UNICHIM על איכות המים לוסיאנה Migliore, Tor Vergata האוניברסיטה (רומא) על שיתוף הפעולה, Rossella Boscolo ו מאסימו Gabellini של Ispra (החוג למניעת הקלה של השפעות) על התמיכה הכספית, פאביו Matassa של לונדון בית הספר לשפות ברומא על התמיכה הלשונית שלהם.

<ol>
	<li>Togulga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Short-Term+Toxicity+of+Two+Toxicants+to+Artemia+Nauplii.">The Short-Term Toxicity of Two Toxicants to Artemia Nauplii.</a> Tr. J. of Zoology. 22, 259-266 (1998).</li><li>Persoone, G., Wells, P. G., Sorgeloos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemia+in+aquatic+toxicology:+a+review.">Artemia in aquatic toxicology: a review.</a> Artemia Research and its Applications. Morphology, Genetics, Strain characterization Toxicology. , 259-275 (1987).</li><li>Nunes, B. S., Carvalho, F. D., Guilhermino, L. M., Stappen, G. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+genus+Artemia+in+ecotoxicity+testing.">Use of the genus Artemia in ecotoxicity testing.</a> Envir. Poll. 144, 453-462 (2006).</li><li>APAT IRSA-CNR. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metodi analitici per le acque Manuali e Linee guida 29/2003. Terzo Metodo, 8060 (2003).</li><li>Sorgellos, P., Wielen, C. R. e. m. i. c. h. e. -. V. a. n. D. e. r., Persoone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Artemia+nauplii+for+toxicity+tests+â€“+a+critical+analysis.">The use of Artemia nauplii for toxicity tests â€“ a critical analysis.</a> Ecotox. Env. Saf. 2, 249-255 (1978).</li><li>Savorelli, F., Palazzi, D., Gorbi, G., Invidia, M., Sei, S., Magaletti, E., Manfra, L., Gelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Set+up+of+a+standard+methodology+for+14-day+bioassay+on+Artemia+franciscana+and+A.+parthenogenetica.">Set up of a standard methodology for 14-day bioassay on Artemia franciscana and A. parthenogenetica.</a> Biol. Amb. 21, 27-36 (2007).</li><li>Brix, K. V., Cardwell, R. D., Adams, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+toxicity+of+arsenic+to+the+Great+Salt+Lake+brine+shrimp,+Artemia+franciscana.">Chronic toxicity of arsenic to the Great Salt Lake brine shrimp, Artemia franciscana.</a> Ecotox. Environ. Saf. 54, 169-175 (2003).</li><li>Brix, K. V., Deforest, D. K., Cardwell, R. D., Adams, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+chronic+site-specific+water+quality+standard+for+selenium+in+the+Great+Salt+Lake,+Utah,+USA.">Derivation of a chronic site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA.</a> Environ. Toxicol. Chem. 23, 606-612 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualit&#225;+dell'acqua+-+Determinazione+della+tossicit&#225;+letale+a+14+giorni+con+Artemia+franciscana+(Crustacea:+Anostraca).">Qualit&#225; dell'acqua - Determinazione della tossicit&#225; letale a 14 giorni con Artemia franciscana (Crustacea: Anostraca).</a> Commissione UNICHIM "Qualit&#225; dell'acqua" Gruppo di Lavoro "Metodi Biologici" Sottogruppo "Acque salate/salmastre e sedimenti". , (2010).</li><li>Varo, I., Navarro, J. C., Amat, F., Guilhermino, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+cholinesterases+and+evaluation+of+the+inhibitory+potential+of+chlorpyrifos+and+dichlorvos+to+Artemia+salina+and+Artemia+parthenogenetica.">Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica.</a> Chem. 48, 563-569 (2002).</li></ol>

אין ניגוד עניינים הצהיר.

הארטמיה הוא אחד היצורים הבדיקה הערך הרב ביותר עבור בדיקות ecotoxicity מחקר שנעשה עד כה מאפשר לנו לקבוע כי אפשר לקיים מספר אפשרויות הקשורות לשימוש הארטמיה ב טוקסיקולוגיה Ecotoxicology. המאפיינים שהופכים את האורגניזם למין מתאימים bioassays הם: פיזור גיאוגרפי רחב, הסתגלות גב...

הופק על ידי Loredana Manfra של מכון המחקר להגנה על הסביבה (Ispra-מחלקת ניטור איכות הסביבה) ו פדריקה Savorelli של הסוכנות האזורית לאיכות הסביבה (Emilia-Romagna, איטליה). התייחסות חברי עבור קבוצת המשנה העבודה על הארטמיה לסרטן בקבוצת שיטות ביולוגיות - מים מלוחים / מים מליחים ומשקעים של ועדת UNICHIM על איכות המים (המכון הפעלה תקינה טכנית במגזר כימי). Videography ועריכה על ידי מרקו Pisapia של Ispra (המדעית המייעצת שירותי תיעוד). תיאום הפקה של אנה מריה קיקרו אריקה Magaletti של Ispra (מחלקת ניטור איכות הסביבה). אנו רוצים להודות הסוכנות האזורית לאיכות הסביבה של אמיליה, רומניה, סניף פרארה Biologקבוצת שיטות iCal - מים מלוחים / מים מליחים ומשקעים של ועדת UNICHIM על איכות המים על שיתוף הפעולה, Rossella Boscolo ו מאסימו Gabellini של Ispra (החוג למניעת הקלה של השפעות) על התמיכה הכספית, ובית הספר בלונדון של שפות ברומא על התמיכה הלשונית שלהם. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר מוצר </td></tr><tr><td> Dodecyl סודיום סולפט </td><td> Sigma-Aldrich </td><td> L4390 </td></tr></tbody></table> נספח התקשורת אצות צריך להיות מוכנים באמצעות ריאגנטים איכות אנליטיות. ליטר של פתרון מזין מוכן ידי המסת 300 גר &#39;3 Nano, 30 גר&#39; גר &#39;קרן היסוד PO 4 ו -20 של 2 Cl 4 NH במים מזוקקים. ארבעת הפתרונות הראשונים (A, B, C, D) של מינרלים מוכנים כדלקמן: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ליטר של פתרון &quot;&quot; המכיל 30 גרם של ZnSO 4 H 2 O, 25 גרם של CuSO 4 5 H 2 O, 30 גרם של COSO 4 7 H 2 O ו - 20 גרם של MnSO 4 H 2 O במים מזוקקים; </li><li style=";text-align:right;direction:rtl"> ליטר של פתרון &quot;B&quot; המכילה 50 גרם של FeCl 3 • 6 H 2 O במים מזוקקים; </li><li style=";text-align:right;direction:rtl"> ליטר של פתרון &quot;C&quot;, המכיל 25 גרם של Na 2 מו 4 • 2 H 2 O במים מזוקקים; </li><li style=";text-align:right;direction:rtl"> ליטר של פתרון &quot;D&quot; המכיל 50 גרם די Na 2 EDTA • 2 H 2 O במים מזוקקים. </li></ul> ליטר של הפתרון הסופי של מינרלים (פתרון &quot;E&quot;) מתקבל על הוספת aliquots של הנ&quot;ל פתרונות מים מזוקקים. לפי הסדר הבא של בנוסף מומלץ: 100 מ&quot;ל של פתרון &quot;D&quot;, 10 מ&quot;ל של פתרון &quot;C&quot;, 10 מ&quot;ל של פתרון &quot;B&quot; ו - 10 מ&quot;ל של פתרון &quot;&quot;. שלושה initiaאני פתרונות (F, G, H) של ויטמינים מוכנים כדלקמן: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ליטר של פתרון &quot;F&quot; המכיל 100 מ&quot;ג של ביוטין במים מזוקקים; </li><li style=";text-align:right;direction:rtl"> ליטר של פתרון &quot;G&quot;, המכיל 10 גרם של תיאמין במים מזוקקים; </li><li style=";text-align:right;direction:rtl"> ליטר של פתרון &quot;H&quot; המכיל 100 מ&quot;ג של cobalamin במים מזוקקים. </li></ul> ליטר של הפתרון הסופי של ויטמינים (פתרון &quot;אני&quot;) מתקבל על הוספת 10 מ&quot;ל של שלושת הפתרונות הראשונים למים מזוקקים. ליטר של מדיה אצות מכינים על ידי המסת 1 מ&quot;ל של פתרון מזין, 1 מ&quot;ל של תמיסת מינרלים האחרון (פתרון &quot;E&quot;) ו - 1 מ&quot;ל של תמיסת ויטמין האחרון (פתרון &quot;אני&quot;) במי ים. האחרון זה ניתן לקבל גם באמצעות המסת חומרים כימיים המוכרים איכות אנליטיות או ניסוח זמינים מסחרית במים מזוקקים או דה מיונן. תערובות מלח ליצירת מים מלוחים אידיאלי זמינים באופן מסחרי, למשל, את תערובות האוקיינוס ​​מיידיות. זה recommended באמצעות מי ים עם מליחות של 30 (± 2) PSU וטמפרטורה של 20 (± 1) ° C.

long-term lethal toxicity test with the crustacean artemia franciscana

פעילויות המחקר שלנו למקד את השימוש בשיטות ביולוגיות לצורך הערכה של איכות הסביבה, תוך התייחסות מיוחדת מים מלוחים / מים מליחים ומשקעים. הבחירה של אינדיקטורים ביולוגיים חייב להיות מבוסס על ידע מדעי אמין, אולי, על זמינותם של נהלים אחידים. במאמר זה, אנו מציגים פרוטוקול סטנדרטי המשמש לסרטן ימית הארטמיה להעריך את רעילות של כימיקלים ו / או של מטריצות הסביבה הימית. המדענים מציעים כי ארטמיה (הארטמיה) הוא המועמד המתאים לפיתוח המבדק תקן ניצול ברחבי העולם. מספר מאמרים פורסמו על ההשפעות הרעילות של כימיקלים שונים על toxicants ארטמיה (הארטמיה). היתרון הגדול של לסרטן זה ללימודים רעילות הוא הזמינות הכללית של ציסטות יבשים אלה יכולים לשמש מיד בבדיקת וטיפוח קשה לא דרש <sup&gt; 1,2. ציסטה על בסיס מבחני רעילות הם זולים, זמינים באופן רציף, פשוטה ואמינה והם ולכן התשובה חשובה לצרכים שגרתיים של ההקרנה רעילות, דרישות ניטור תעשייתיים או למטרות רגולטוריות 3. השיטה המוצעת כרוך בתמותה כנקודת הסיום. המספרים של ניצולי נספרו ואחוז מקרי מוות חושבו. הזחלים נחשבו מתים אם הם לא מפגינים כל תנועה פנימית או חיצונית במהלך כמה שניות של התבוננות 4. הליך זה היה בדיקה סטנדרטית חומר ייחוס (Dodecyl נתרן גופרתי), כמה תוצאות מדווחים עבודה זו. מאמר זה מלווה בסרטון המתאר את ביצוע בדיקות רעילות פרוצדורלי, מראה את כל השלבים הקשורים בפרוטוקול.

Watch this Scientific Journal Video about Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana at JoVE.com

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

מחקר זה עוסק בפיתוח סטנדרטיזציה של פרוטוקול מתודולוגית חשוב לקבוע לטווח ארוך (14 יום) רעילות קטלני המופעל על ידי חומרים כימיים, שפכים תעשייתיים או ביוב דגימות סביבתיות נוזליים על לסרטן מים מלוחים,<em&gt; הארטמיה franciscana</em&gt;.

לטווח ארוך רעילות מבחן קטלני עם לסרטן הארטמיה franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

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Biology

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

19.4K Views.  Institute for Environmental Protection and Research. מחקר זה עוסק בפיתוח סטנדרטיזציה של פרוטוקול מתודולוגית חשוב לקבוע לטווח ארוך (14 יום) רעילות קטלני המופעל על ידי חומרים כימיים, שפכים תעשייתיים או ביוב דגימות סביבתיות נוזליים על לסרטן מים מלוחים, הארטמיה franciscana.

Video: Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

לטווח ארוך הדמיה בתאי יונקים באמצעות מיקרוסקופית שדה רחב

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

לטווח ארוך של תרבות האדם סרטן השד דוגמאות וניתוח שלהם באמצעות טומוגרפיה הקרנה אופטית

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

לטווח ארוך הדמיה, ברזולוציה גבוהה Confocal זמן פקיעה של<em&gt; טבורית ארבידופסיס</em&gt; אפידרמיס במהלך הנביטה

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

רב שכבתי וההרכבה למיקרוסקופיה גיליון אור לטווח ארוך של דג הזברה

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA, שיטה להרכבה ממושכת על זחל לטווח ארוך הדמיה לזמן קצר

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

בידוד, טרנס-תירבה ותרבות ארוכת טווח של עכבר מבוגר וקרדיומיוציטים עכברים וחולדות

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

יישום של תנועת ראש פסיבית ליצירת תאוצות מוגדרות בראשי מכרסמים

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

איסוף ותחזוקה ארוכת טווח של נמלים חותכות עלים (Atta) בתנאי מעבדה

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

תרבות ארוכת טווח של Caenorhabditis elegans בודדים על מדיה מוצקה לניטור פלואורסצנטי אורכי והתערבויות מרתיעות

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

תרבית ארוכת טווח וניטור של Caenorhabditis elegans מבודדים על מדיה מוצקה במכשירים מרובי בארות

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...