Name: preparation of drosophila larval samples for gas chromatography-mass spectrometry (gc-ms)-based metabolomics
Uploaded: 2018-06-06T15:00:00.000+00:00
Duration: 7 min 21 s
Description: Watch this Scientific Journal Video about Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics at JoVE.com

תודה לחברים של המתקן ספקטרוסקופיית מסה אוניברסיטת אינדיאנה, המתקן הליבה גליקומיקס אוניברסיטת יוטה לעזרה בארגון אופטימיזציה של פרוטוקול זה. J.M.T. נתמך על ידי הלאומית המכון כללי רפואי למדעים של מכוני הבריאות הלאומיים תחת פרס מספר R35GM119557.

<ol>
	<li>Owusu-Ansah, E., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+metabolic+homeostasis+and+nutrient+sensing+in+Drosophila:+implications+for+aging+and+metabolic+diseases.">Modeling metabolic homeostasis and nutrient sensing in Drosophila: implications for aging and metabolic diseases.</a> Disease Models &amp; Mechanisms. 7 (3), 343-350 (2014).</li><li>Sieber, M. H., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+metabolic+states+in+development+and+disease.">The role of metabolic states in development and disease.</a> Current Opinion in Genetics &amp; Development. 45, 58-68 (2017).</li><li>Tennessen, J. M., Barry, W. E., Cox, J., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68 (1), 105-115 (2014).</li><li>Li, H., 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=Drosophila+larvae+synthesize+the+putative+oncometabolite+L-2-hydroxyglutarate+during+normal+developmental+growth.">Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (6), 1353-1358 (2017).</li><li>Tennessen, J. M., Baker, K. D., Lam, G., Evans, J., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+Estrogen-Related+Receptor+Directs+a+Metabolic+Switch+that+Supports+Developmental+Growth.">The Drosophila Estrogen-Related Receptor Directs a Metabolic Switch that Supports Developmental Growth.</a> Cell Metabolism. 13 (2), 139-148 (2011).</li><li>Nicholson, J. K., Lindon, J. C., Holmes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabonomics':+understanding+the+metabolic+responses+of+living+systems+to+pathophysiological+stimuli+via+multivariate+statistical+analysis+of+biological+NMR+spectroscopic+data.">Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data.</a> Xenobiotica. 29 (11), 1181-1189 (1999).</li><li>Fiehn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+-+the+link+between+genotypes+and+phenotypes.">Metabolomics - the link between genotypes and phenotypes.</a> Plant Molecular Biology. 48 (1-2), 155-171 (2002).</li><li>Cox, J. E., Thummel, C. S., Tennessen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+Studies+in+Drosophila.">Metabolomic Studies in Drosophila.</a> Genetics. 206 (3), 1169-1185 (2017).</li><li>Lenz, E. M., Wilson, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+strategies+in+metabonomics.">Analytical strategies in metabonomics.</a> Journal of Proteome Research. 6 (2), 443-458 (2007).</li><li>Pasikanti, K. K., Ho, P. C., Chan, E. C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatography/mass+spectrometry+in+metabolic+profiling+of+biological+fluids.">Gas chromatography/mass spectrometry in metabolic profiling of biological fluids.</a> Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. 871 (2), 202-211 (2008).</li><li>Want, E. J., Nordstrom, A., Morita, H., Siuzdak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+exogenous+to+endogenous:+The+inevitable+imprint+of+mass+spectrometry+in+metabolomics.">From exogenous to endogenous: The inevitable imprint of mass spectrometry in metabolomics.</a> Journal of Proteome Research. 6 (2), 459-468 (2007).</li><li>Garcia, A., Barbas, C., Metz, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metabolic Profiling: Methods and Protocols Vol. 708 Methods in Molecular Biology. , 191-204 (2011).</li><li>Chan, E. C. Y., Pasikanti, K. K., Nicholson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+urinary+metabolic+profiling+procedures+using+gas+chromatography-mass+spectrometry.">Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry.</a> Nature Protocols. 6 (10), 1483-1499 (2011).</li><li>Dunn, W. B., 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=Procedures+for+large-scale+metabolic+profiling+of+serum+and+plasma+using+gas+chromatography+and+liquid+chromatography+coupled+to+mass+spectrometry.">Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry.</a> Nature Protocols. 6 (7), 1060-1083 (2011).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Laboratory Manual. , 171-178 (1989).</li><li>Biyasheva, A., Do, T. V., Lu, Y., Vaskova, M., Andres, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glue+secretion+in+the+Drosophila+salivary+gland:+a+model+for+steroid-regulated+exocytosis.">Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis.</a> Developmental Biology. 231 (1), 234-251 (2001).</li><li>Lommen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetAlign:+Interface-driven,+versatile+metabolomics+tool+for+hyphenated+full-scan+mass+spectrometry+data+preprocessing.">MetAlign: Interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing.</a> Analytical Chemistry. 81 (8), 3079-3086 (2009).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+MetaboAnalyst+3.0+for+comprehensive+metabolomics+data+analysis.">Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis.</a> Current Protocols in Bioinformatics. 55, (2016).</li><li>Xia, J., Sinelnikov, I. V., Han, B., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetaboAnalyst+3.0-making+metabolomics+more+meaningful.">MetaboAnalyst 3.0-making metabolomics more meaningful.</a> Nucleic Acids Research. 43 (W1), W251-W257 (2015).</li><li>Lommen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+(pre-)processing+of+nominal+and+accurate+mass+LC-MS+or+GC-MS+data+using+MetAlign.">Data (pre-)processing of nominal and accurate mass LC-MS or GC-MS data using MetAlign.</a> Methods in Molecular Biology. 860, 229-253 (2012).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+MetaboAnalyst+3.0+for+comprehensive+metabolomics+data+analysis.">Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis.</a> Current Protocols in Bioinformatics. 55, (2016).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web-based+inference+of+biological+patterns,+functions+and+pathways+from+metabolomic+data+using+MetaboAnalyst.">Web-based inference of biological patterns, functions and pathways from metabolomic data using MetaboAnalyst.</a> Nature Protocols. 6 (6), 743-760 (2011).</li><li>Li, H., Tennessen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+the+metabolic+basis+of+Drosophila+development.">Methods for studying the metabolic basis of Drosophila development.</a> Wiley Interdisciplinary Reviews Developmental Biology. 6 (5), (2017).</li></ol>

המחברים אין לחשוף.

גליקומיקס מספק הזדמנות ללא תחרות סקר את תגובות חילוף החומרים שמרכיבים את חילוף החומרים מתווך. הרגישות של טכנולוגיה זו, מעבד עם זאת, נתונים רגישים הרקע הגנטי, רמזים התפתחותית, ומגוון של לחצים סביבתיים, לרבות טמפרטורה, לחות, צפיפות אוכלוסין וזמינות חומר מזין. לכן, גבוהה איכות, לשחזור גליקומי?...

זבוב הפירות דרוזופילה melanogaster התפתחה מערכת אידיאלי ללמוד את מנגנון מולקולרי שבו לווסת את חילוף החומרים מתווך. לא רק מסלולים מטבוליים רוב נשמרים דרוזופילה בין בני אדם, אלא מפתח חיישנים התזונתיים ואת הרגולטורים הצמיחה, כגון אינסולין, טור myc, פעילים גם לעוף1,2. כתוצאה מכך, דרוזופילה יכול לשמש כדי לחקור את הבסיס מטבולית של מחלות האדם ועד סוכרת והשמנה הקשורים ניוון מוחיים וסרטן. בהקשר זה, התפתחות הזחל דרוזופילה מספק את המסגרת אידיאלי שבו ללמוד תוכנית מטבולית גליקוליזה אירובי, או ורבורג. בדיוק כמו גידולים רבים להשתמש גליקוליזה אירובי כדי לייצר ביומסה של פחמימות, אז לעשות דרוזופילה הזחלים להפעיל גליקוליזה אירובי כדי לקדם צמיחה התפתחותית-3,-4,-5. אלה קווי דמיון בין זחל ואת חילוף החומרים הגידול להקים דרוזופילה כמו דוגמנית מפתח להבנה כיצד אירובי גליקוליזה הוא מוסדר ויוו.
למרות העובדה כי הזבוב התפתחה כמודל פופולרי עבור הלומדים את חילוף החומרים, רוב המחקרים דרוזופילה להסתמך על שיטות שנועדו למדוד מטבוליטים בודדים3, כגון טרהלוז, טריגליצרידים או ATP. מאז פרוטוקול מסוים נדרש כדי למדוד כל מטבוליט, מחקרים מבוססי assay הם עתירי עבודה, יקר, מוטה לכיוון את התרכובות הללו, שניתן למדידה באמצעות ערכות מסחריות. פתרון מגבלות אלה התפתחה מן התחום גליקומיקס, אשר מספק אמצעים יותר יעיל, לא משוחד של הלומדים דרוזופילה חילוף החומרים. בניגוד מחקר מבוסס-assay, ניתוח metabolomic יחיד יכול בו זמנית של מולקולה קטנה מטבוליטים מאות וכולן מספקות והיכרות מעמיקה של אורגניזם מצב מטבולי6,7. טכניקה זו התרחב באופן משמעותי את היקף לימודי מטבולית דרוזופילה ומייצג את העתיד של שדה המתעוררים זה8.
Metabolomic הלימודים מתנהלים בעיקר באמצעות שלוש טכנולוגיות: (i) תהודה מגנטית גרעינית (NMR), כרומטוגרפיה נוזלית (ii) מסות (LC-MS) ו- (iii) גז כרומטוגרפיה-ספקטרומטריית (GC-MS)9. בכל אחת מהגישות מציע יתרונות וחסרונות, ו כל הטכנולוגיות האלה שימשו ללמוד בהצלחה דרוזופילה חילוף החומרים. מאז המחקרים שנערכו במעבדה שלנו מתמקדת מטבוליטים קטן, קוטב, אנו מעסיקים בעיקר בשיטה מבוססת-GC-MS. GC-MS מספק למשתמש עם מספר רב של יתרונות, כולל הפארמצבטית גבוהה, שיא רזולוציה, רגישות, וגילה הזמינות של ספרייה ספקטרלי ההשפעה (EI) אלקטרון רגיל, המאפשר זיהוי מהיר של חילוף החומרים תכונות10,11. ההכנה של דגימות GC-MS, אולם, היא מורכבת למדי, דורש את תשומת לב רבה לפרטים. דוגמאות חייב להיות שנאספו, שטף, שקל, קפוא באופן במהירות המרווה תגובות חילוף החומרים. יתר על כן, הגופה לטוס עמיד בפני פרוטוקולים סטנדרטיים המגון ודורש טחנה חרוז כדי להבטיח מיצוי מטבוליט אופטימלית. לבסוף, דגימות נותחו על ידי GC-MS חייב לעבור derivatization כימי לפני זיהוי12. בעוד שיטות שפורסמו בעבר לתאר כל אלה13,3,השלבים14, פרוטוקול חזותי אשר יאפשרו למשתמש המתחיל להפיק reproducibly נתוני איכות גבוהה עדיין נדרשת. כאן נדגים כיצד להתכונן גליקומיקס GC-MS מבוססי ניתוח דגימות זחל דרוזופילה . פרוטוקול זה מאפשר למשתמש למדוד רבים של מטבוליטים קוטב קטנים שמרכיבים פחמן מרכזי חילוף החומרים reproducibly.

<table><tbody><tr><td>Unsulfured blackstrap molasses</td><td>Good Food, INC</td><td></td><td></td></tr><tr><td>Drosophila Agar Type II</td><td>Genesee Scientific</td><td>66-103</td><td></td></tr><tr><td>Pyridine</td><td>EMD Millipore</td><td>PX2012-7</td><td></td></tr><tr><td>Methoxyamine hydrocholoride (MOX)</td><td>MP Biomedicals, LLC</td><td>155405</td><td></td></tr><tr><td>MSTFA with 1% trimethylchlorosilane</td><td>Sigma</td><td>69478</td><td></td></tr><tr><td>Fleischmann&amp;rsquo;s Active dry yeast</td><td>AB Mauri Food Inc</td><td>2192</td><td></td></tr><tr><td>6oz Drosophila stock bottle</td><td>Genesee Scientific</td><td>32-130</td><td></td></tr><tr><td>Soft tissue homogenizing mix (2 mL tubes)&amp;nbsp;</td><td>Omni International</td><td>SKU:19-627</td><td></td></tr><tr><td>Vial insert, 250 &amp;micro;L deactivated glass with polymer feet</td><td>Agilent</td><td>5181-8872</td><td></td></tr><tr><td>Succinic acid-2,2,3,3-d&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>293075</td><td></td></tr><tr><td>SpeedVac</td><td>Thermo&amp;nbsp;</td><td>SC210A</td><td></td></tr><tr><td>o-Phosphoric acid</td><td>Fisher Scientific</td><td>A242-1</td><td></td></tr><tr><td>Propionic acid</td><td>Sigma</td><td>P5561</td><td></td></tr><tr><td>p-Hydroxy benzoic acid methyl ester</td><td>Genesee Scientific</td><td>20-258</td><td></td></tr><tr><td>Bead Ruptor</td><td>Omni International</td><td>SKU:19-040E</td><td></td></tr><tr><td>ThermoMixer F1.5</td><td>Eppendorf</td><td>5384000012</td><td></td></tr><tr><td>MultiTherm Shaker with a 24 X 12 mm block</td><td>Benchmark Scientific</td><td>H5000</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td></td></tr><tr><td>1.5 mL centrifuge tube</td><td>Eppendorf</td><td>22364111</td><td></td></tr><tr><td>Falcon 35 X 10 mm tissue culture dish</td><td>Corning Incorporated</td><td>353001</td><td></td></tr><tr><td>GC column</td><td>Phenomex</td><td>ZB-5MSi</td><td></td></tr></tbody></table>

preparation of drosophila larval samples for gas chromatography-mass spectrometry (gc-ms)-based metabolomics

ההתקדמות בתחום גליקומיקס הקימו זבוב הפירות דרוזופילה melanogaster כמודל גנטי רב עוצמה עבור הלומדים חילוף החומרים בעלי חיים. על ידי שילוב המערך העצום של כלים גנטיים דרוזופילה עם היכולת סקר כ"שטח נוף פתוח של חילוף החומרים מתווך, בגישה גליקומיקס יכול לחשוף לאינטרקציות מורכבות בין דיאטה, גנוטיפ, אירועי חיים-היסטוריה רמזים סביבתיים. בנוסף, מחקרים גליקומיקס ניתן לגלות מנגנונים אנזימטי הרומן, לחשוף קשרים ידועים בין מסלולים מטבוליים שונים לכאורה. על מנת להקל על השימוש הנרחב יותר של טכנולוגיה זו בקרב הקהילה דרוזופילה , כאן אנחנו מספקים. פרוטוקול מפורט המתאר כיצד להתכונן דגימות זחל דרוזופילה ספקטרומטריית גז כרומטוגרפיה (GC-MS)- metabolomic המבוססת על ניתוח. פרוטוקול שלנו כולל תיאורים של איסוף הדגימה זחל, מטבוליט החילוץ, derivatization כימי וניתוח GC-MS. סיומו המוצלח של פרוטוקול זה יאפשר למשתמשים למדוד את השפע היחסי של מטבוליטים קוטב קטנים, כולל חומצות אמינו, סוכרים, חומצות אורגניות מעורב גליקוליזה את מחזורי TCA.

לקטט דהידרוגנאז מוטציות (dLDH), שחסרה פעילות dLDH4ופקדים מתאימים מבחינה גנטית נאספו כ אמצע-L2 הזחלים ומעובדים על-פי הפרוטוקול המתואר לעיל. בהשוואה עם פקדים, הזחלים מוטציה התערוכה שינויים משמעותיים לקטט פירובט, L-2-hydroxyglutarate4. ספקטרה נרכשו עם מערכת A...

Watch this Scientific Journal Video about Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics at JoVE.com

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

פרוטוקול זה מתאר כיצד להתכונן הרימות דרוזופילה metabolomic GC-MS מבוססי ניתוח.

הכנת דוגמאות זחל דרוזופילה עבור גז כרומטוגרפיה-ספקטרומטריית (GC-MS)-מבוסס מטבולומיקס

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

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JoVE Journal

Developmental Biology

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

11.1K Views.  Indiana University. פרוטוקול זה מתאר כיצד להתכונן הרימות דרוזופילה metabolomic GC-MS מבוססי ניתוח.

Video: Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

הכנת הברים מועשר מיטוכונדריאלי לניתוח חילוף חומרים ב<em&gt; דרוזופילה</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

הכנה בינונית של<em&gt; תסיסנית</em&gt; עוברי תמציות עבור ניסויים פרוטאומיים

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

חיתוך של דרוזופילה מלאנוסטר שרירי הטיסה לגישות omics

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

Genetics

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

למידה אסוציאטיבית הרחה באפטטיבית<em&gt; דרוזופילה</em&gt; זחלים

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Neuroscience

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

הכנת לפתח רקמות היקפיים חוש הריח עבור מולקולרית וניתוח Immunohistochemical ב דרוזופילה

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

<meta charset="utf8"></head><body>פלטפורמה "תוצרת בית" משתלמת ויעילה למחקרי התנהגות של דרוזופילה, ופרוטוקול נלווה לרספירומטריה מיטוכונדריאלית של זחלים

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

פלטפורמה "תוצרת בית" משתלמת ויעילה למחקרי התנהגות של דרוזופילה , ופרוטוקול נלווה לרספירומטריה מיטוכונדריאלית של זחלים

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

Behavior

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

<meta charset="utf8"></head><body>ניתוח איתות שומנים בקולטני אור דרוזופילה באמצעות ספקטרומטריית מסה

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

ניתוח איתות שומנים בקולטני אור דרוזופילה באמצעות ספקטרומטריית מסה

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Biochemistry

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

ניתוח מטבולית של melanogaster דרוזופילה Larval ושכל למבוגרים

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

הכנת דוגמאות זחל דרוזופילה עבור גז כרומטוגרפיה-ספקטרומטריית (GC-MS)-מבוסס מטבולומיקס

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הכנת הברים מועשר מיטוכונדריאלי לניתוח חילוף חומרים ב

הכנה בינונית של

חיתוך של דרוזופילה מלאנוסטר שרירי הטיסה לגישות omics

למידה אסוציאטיבית הרחה באפטטיבית

הכנת לפתח רקמות היקפיים חוש הריח עבור מולקולרית וניתוח Immunohistochemical ב דרוזופילה

פלטפורמה "תוצרת בית" משתלמת ויעילה למחקרי התנהגות של דרוזופילה , ופרוטוקול נלווה לרספירומטריה מיטוכונדריאלית של זחלים

פלטפורמה "תוצרת בית" משתלמת ויעילה למחקרי התנהגות של דרוזופילה , ופרוטוקול נלווה לרספירומטריה מיטוכונדריאלית של זחלים

ניתוח איתות שומנים בקולטני אור דרוזופילה באמצעות ספקטרומטריית מסה

ניתוח איתות שומנים בקולטני אור דרוזופילה באמצעות ספקטרומטריית מסה

ניתוח מטבולית של melanogaster דרוזופילה Larval ושכל למבוגרים