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>
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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 border="0" cellpadding="0" cellspacing="0" style="width:827px;" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Unsulfured blackstrap molasses</td>
			<td style="width:141px;">Good Food, INC</td>
			<td style="width:141px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td>Genesee Scientific</td>
			<td>66-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyridine</td>
			<td>EMD Millipore</td>
			<td>PX2012-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methoxyamine hydrocholoride (MOX)</td>
			<td>MP Biomedicals, LLC</td>
			<td>155405</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MSTFA with 1% trimethylchlorosilane</td>
			<td>Sigma</td>
			<td>69478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fleischmann&#8217;s Active dry yeast</td>
			<td>AB Mauri Food Inc</td>
			<td>2192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6oz Drosophila stock bottle</td>
			<td>Genesee Scientific</td>
			<td>32-130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soft tissue homogenizing mix (2 mL tubes)&#160;</td>
			<td>Omni International</td>
			<td>SKU:19-627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vial insert, 250 &#181;L deactivated glass with polymer feet</td>
			<td>Agilent</td>
			<td>5181-8872</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Succinic acid-2,2,3,3-d4</td>
			<td>Sigma</td>
			<td>293075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpeedVac</td>
			<td>Thermo&#160;</td>
			<td>SC210A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">o-Phosphoric acid</td>
			<td>Fisher Scientific</td>
			<td>A242-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propionic acid</td>
			<td>Sigma</td>
			<td>P5561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-Hydroxy benzoic acid methyl ester</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead Ruptor</td>
			<td>Omni International</td>
			<td>SKU:19-040E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ThermoMixer F1.5</td>
			<td>Eppendorf</td>
			<td>5384000012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MultiTherm Shaker with a 24 X 12 mm block</td>
			<td>Benchmark Scientific</td>
			<td>H5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL centrifuge tube</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35 X 10 mm tissue culture dish</td>
			<td>Corning Incorporated</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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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Developmental Biology

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

11.0K 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 ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

דיסקציה של רקמות הזחל דג הזברה האשכים

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

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, ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

הקמתה של הזחל דג זברה כמודל חיה כדי Investigate Trypanosoma cruzi תנועתיות Vivo ב

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

מפשיר Culturing, Cryopreserving שורות תאים דרוזופילה

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

שיטת שלמות המוח בדם בדרוזופילה מלאנוסטר

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

בדיקת גודל הכבד בזחל זברה באמצעות מיקרוסקופ ברייטפילד

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

פיתוח מודל לזיהום של זחל בדג במשך שינוי קושי

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

הכנת מדרוזופילה זחל ופופל אשכים לניתוח חטיבת תאים בשידור חי, רקמה שלמה

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

דיסקציה, אימונוהיסטוכימיה והרכבה של מוחות זחלים ודרוזופילה בוגרים להדמיית האונה האופטית

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...