עבודה זו נתמכה על ידי פרס טייס ממרכז הלם נתן (P30 AG13283) ומענק R01 (R01 AG028516) מן המכון הלאומי להזדקנות (NIA) ל-A-LH

<html> 1. הקמת עקומת כיול עבור להערכת ריכוזי חיידקים סעיף זה מתאר שיטה להערכת ריכוז החיידקים עבור כל זן חיידקי מסוים (למשל OP50, HT115) אשר ישמשו כמקור מזון עבור ניסויים הגבלה תזונתיים ב C. elegans. עקומות כיול נפרדת יש להקים לכל זן חיידקים המשמשים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחסן מושבה אחת של E. coli OP50 חיידקים שנקטפו פס חדש של חיידקים על אגר LB LB במרק 3 מ&quot;ל נוזל. </li><li style=";text-align:right;direction:rtl"> OP50 המקום והתרבות 37 ° C שייקר ולאפשר לחיידקים לגדול בין לילה. </li><li style=";text-align:right;direction:rtl"> הכנת סידורי 2 פי דילולים של ההשעיה חיידקים מתרבות הלילה (2x, 4x, 8x, 16x, 32x). </li><li style=";text-align:right;direction:rtl"> מדוד את ספיגת (צפיפות אופטית) של דילול זה ב 600 ננומטר על ידי ספקטרופוטומטר בשלושה עותקים. </li><li style=";text-align:right;direction:rtl"> יתר על כן לדלל כל השעיה חיידקי 1,000,000 פי (1000 פי דילול פעמיים). </li><li style=";text-align:right;direction:rtl"> מורחים 0.4 מ&quot;ל של חיידקי ההשעיה של דילול באופן שווה על כל צלחת LB 100 מ&quot;מ אגר. </li><li style=";text-align:right;direction:rtl"> המקום צלחות LB עם OP50 ב 37 ° C חממה 18-24 שעות. </li><li style=";text-align:right;direction:rtl"> ספירת מושבות חיידקים גדל על כל צלחת. ספירת המושבה משמשים כדי לחשב CFU (יחידת המושבה להרכיב) לכל מ&quot;ל עבור כל דגימה. </li><li style=";text-align:right;direction:rtl"> הקשר בין מדד OD 600 ערכים OP50 ריכוזי חיידקים ניתן הוקמה על ידי התוויית OD 600 כפונקציה של ריכוז חיידקי (CFU / mL) ואת ביצוע ניתוח רגרסיה ליניארית (איור 1). </li></ol> פתרונות: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="2"> מרק לוריא (LB), 1 ליטר: </td></tr><tr><td > 10 גרם </td><td> Bacto-tryptone </td></tr><tr><td> 5 גרם </td><td> תמצית שמרים </td></tr><tr><td> 10 גרם </td><td> NaCl </td></tr></tbody></table> מוסיפים מים כדי deionized 1L. החיטוי ולאחסן בטמפרטורת החדר. 2. הכנת מוצק צלחת מבוססי (SDR) צלחות תזונתיים הגבלה סעיף זה מתאר את הכנת צלחות לשימוש מוצק צלחת מבוסס (SDR) ניסויים תזונתיים הגבלה. בדרך כלל, אנו רואים ריכוז חיידקים של 1 x 10 11 CFU / mL כתנאי כרצונך מודעה האכלה 1 x 10 8 CFU / mL כתנאי DR אופטימלי האכלה עבור שיטה זו. עם זאת, 4-6 ריכוזי חיידקים שונים ייתכן שיהיה צורך ליצור מערכת יחסים שלמה מינון תלויי עבור SDR ותגובות שלה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחסן מושבה אחת של E. קולי OP50 חיידקים 3 מ&quot;ל מרק נוזלי LB. </li><li style=";text-align:right;direction:rtl"> מניחים את תרבות OP50 ב 37 ° C שייקר ולאפשר לחיידקים לגדול במשך 6-8 שעות. </li><li style=";text-align:right;direction:rtl"> העברת התרבות OP50 כדי בבקבוק חדש המכיל 500 מ&quot;ל מרק LB, והמקום תרבות OP50 בחזרה 37 ° C שייקר לילה. </li><li style=";text-align:right;direction:rtl"> מדוד את OD 600 של התרבות OP50 כדי לקבוע את ריכוז החיידקים (ריכוז מחושב באמצעות עקומת כיול שנוצר חלק 1). דילולים מתאים עשוי להיות נחוץ עבור מדידה מדויקת של ספיגת. </li><li style=";text-align:right;direction:rtl"> גלולה OP50 חיידקים על ידי צנטריפוגה ב 1500 גרם במשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> Resuspend חיידקים בריכוז של 1 x 10 12 CFU / mL. </li><li style=";text-align:right;direction:rtl"> הכן 10 מ&quot;ל התרבות חיידקים בשישה ריכוזים שונים: 1 x 10 12, 10 11, 10 10, 10 9, 10, 8 10 7 CFU / ml. </li><li style=";text-align:right;direction:rtl"> פיפטה 0.2 מ&quot;ל של התרבות חיידקים למרכז מ&quot;מ 60 הקרושה צלחת NGM. הימנע מנגיעה את פני השטח של צלחת עם פיפטה קצה, כמו ניקס על פני השטח מאפשרים תולעים להתחפר לתוך צלחות אגר. </li><li style=";text-align:right;direction:rtl"> המקום צלחות 37 ° C החממה במשך שעה 1. </li><li style=";text-align:right;direction:rtl"> החל 10μL של 100mg/mL carbenicillin ו 10 μL של kanamycin 50mg/mL על כל צלחת לעצור את הצמיחה של החיידקים. </li><li style=";text-align:right;direction:rtl"> חנות צלחות עם ריכוזים שונים של אנטיביוטיקה, הרג חיידקים על 4 מעלות צלזיוס לשימוש עתידי (עד חודש). חשוב להכין ולאחסן צלחות מספיק הניסוי המתוכנן כולו לפחות 1-2 ימים קדימה. באמצעות אצווה זהה של הצלחות הניסויים כולו עדיפה. </li><li style=";text-align:right;direction:rtl"> כדי לבדוק את הכדאיות של חיידקים, חיידקים לגרד כמה מתוך צלחות SDR ופרש אותה על צלחת ריקה NGM עם carbenicillin ו kanamycin. מניחים את צלחת 37 ° C בחממה במשך 6-8 שעות ולאחר מכן לבדוק צמיחה של חיידקים (אין צמיחה חיידקים יש לשים לב). </li></ol> פתרונות <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="2"> צמיחה נמטודות מדיה (NGM), 1 ליטר: </td></tr><tr><td > 3 גרם </td><td> NaCl </td></tr><tr><td> 2.5 גרם </td><td> Bacto-Peptone </td></tr><tr><td> 20 גרם </td><td> אגר </td></tr></tbody></table>ove_content &quot;&gt; החיטוי במשך 40 דקות ולתת להתקרר 65 מעלות צלזיוס, ולאחר מכן להוסיף: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td > 1 מ&quot;ל </td><td> 1 מ 4 MgSO </td></tr><tr><td> 1 מ&quot;ל </td><td> 1 M CaCl 2 </td></tr><tr><td> 1 מ&quot;ל </td><td> 5 מ&quot;ג / מ&quot;ל ​​כולסטרול </td></tr><tr><td> 25 מ&quot;ל </td><td> אשלגן פוספט 1M, 6.0 pH </td></tr></tbody></table> מיד NGM לשפוך נוזל על 60 מ&quot;מ x 15 מ&quot;מ בצלחות פטרי ב 10 מ&quot;ל aliquots 3. הגדרת ניסויים SDR עבור רוב SDR-הקשורים ללימודים, כגון ניתוח תוחלת החיים ואת פרופיל פוריות, הכנת האוכלוסייה בגיל מסונכרן של תולעים היא חיונית. עבור מחקרים אחרים, פשוט להעביר תולעים מצלחת לצלחת OP50 קבוע SDR כדי ליזום את הגבלת תזונתיים. בסעיף זה, פרוטוקול להקים ניתוח תוחלת החיים של בעלי חיים SDR מתוארת כדוגמה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש בורר תולעת פלטינה להעביר 20-30 מבוגרים reproductively פעיל על מספר הצלחות NGM קבוע עם OP50. בהתאם למספר הביצים הדרוש הניסויים, מבוגרים יותר עשוי להיות נחוץ. </li><li style=";text-align:right;direction:rtl"> השאירו את הצלחות בטמפרטורה של 20 מעלות צלזיוס למשך 4 שעות כדי לאפשר ההטלה. </li><li style=";text-align:right;direction:rtl"> הסר את המבוגרים מן הצלחת. צלחות יש לבדוק ויזואלית את הביצים. </li><li style=";text-align:right;direction:rtl"> השאירו את הצלחות בטמפרטורה של 20 מעלות צלזיוס ולאפשר הצאצאים לפתח לשלב Late-L4/day 1 מבוגר. בדרך כלל זה לוקח 3 ימים תולעים N2 סוגי בר ב 20 ° C. </li><li style=";text-align:right;direction:rtl"> העברת מספר מתאים של 1 מבוגרים יום לצלחת SDR כדי ליזום את הגבלת תזונתיים. </li><li style=";text-align:right;direction:rtl"> לניתוח תוחלת החיים, להקים סך של 6-8 צלחות עם 10-20 תולעים לכל צלחת עבור כל תנאי SDR. </li><li style=";text-align:right;direction:rtl"> ציון את הכדאיות של כל תולעת כל 2 ימים עד שכל התולעים מתו. זה קריטי, כי תולעים יועברו צלחות SDR טריים כל 12-48 שעות כדי למנוע קיפוח מוחלט של מזון. </li></ol> 4. נציג תוצאות: שמוצג באיור 1 היא מזימה של עקומת כיול עבור נציג להערכת ריכוז OP50 ביחס OD 600. המשוואה שנוצר על ידי ניתוח רגרסיה אניה יכול לשמש עבור כל החישובים העתיד של OP50 ריכוזי חיידקים. שמוצג באיור 2 הוא דוגמה assaying את ההשפעה של טיפולים SDR כרונית על תוחלת החיים הממוצעת של זנים שונים של תולעים. הקו הכחול מייצג תגובה נורמלית מינון תלויי של wild-type תולעים SDR. הקו האדום מציין כי ההשפעה של תוחלת החיים SDR מודחק חלקית על ידי drr OE-2, בעוד daf-16 (mu86) אין כל השפעה על הארכת תוחלת החיים SDR-Induced. פירוט השיטה לביצוע ניתוח תוחלת החיים ב C. elegans מתואר בספרות 1-3 הקודמת. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="איור 1" /> באיור 1. התוצאה נציג של עקומת כיול עבור להערכת ה OP50 קולי ריכוז. OP50 ריכוז זממו כפונקציה של מדד OD 600 ערכים. ברים שגיאה = התרבות חיידקים SD לילה היה מדולל 8x 1.5x, 2x, 4x, 16x, 32x לפני מדידת ספיגת ו בדילול עוד 1,000 x פעמיים לפני המדידה CFU. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="איור 2" /> איור 2. התוצאה נציג של C. הניסוי elegans תוחלת החיים השוואת N2 סוג חיות בר (כחול) עם שתי מוטציות שונות (ירוק ואדום) בתגובה SDR (איור מתוך צ&#39;ינג et al. 4). ברים שגיאה = SEM

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הגבלה תזונתיים (DR) ידוע להגדיל את תוחלת החיים ואת לעכב את התחלתה של מחלות הקשורות לגיל שונים במגוון רחב של מינים, כולל יונקים 5-7. מניפולציות הן סביבתיים וגנטיים שימשו מודל DR ב C. elegans, כולל תולעים culturing במדיום למחצה מוגדרים נוזל 8,9, דילול של מקור מזון חייד...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> Carbenicillin </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> מומס במים. </td></tr><tr><td> Kanamycin </td><td> רוש </td><td> 10106801001 </td><td> מומס במים. </td></tr><tr><td> Bacto-peptone </td><td> בקטון, דיקינסון החברה </td><td> DF0118072 </td><td></td></tr><tr><td> Bacto-tryptone </td><td> בקטון, דיקינסון החברה </td><td> DF0123075 </td><td></td></tr><tr><td> Bacto-תמצית שמרים </td><td> בקטון, דיקינסון החברה </td><td> DF0127071 </td><td></td></tr><tr><td> אגר </td><td> בקטון, דיקינסון החברה </td><td> DF00145170 </td><td></td></tr><tr><td> תרבות סטרילי צינור </td><td> ארה&quot;ב מדעי </td><td> 1485-0810 </td><td></td></tr><tr><td> ספקטרופוטומטר </td><td> Amersham Biosciences </td><td> ג&#39;ין קוואנט הפרו </td><td> Amersham הוא כעת חלק GE Healthcare. </td></tr><tr><td> הביתור מיקרוסקופ </td><td> Zeiss </td><td> STEMI 2000 </td><td></td></tr><tr><td> סרכזת </td><td> Beckman Coulter </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

הפחתת צריכת המזון ללא רעב או תת תזונה ידוע להגדיל את תוחלת החיים ואת לעכב את התחלתה של מחלות הקשורות לגיל שונים במגוון רחב של מינים, כולל יונקים. זה גם גורם לירידה במשקל הגוף, פוריות, כמו גם רמות נמוכות של גלוקוז בדם, אינסולין, IGF-1 אצל בעלי חיים אלה. טיפול זה המכונה לעתים קרובות מגבלה תזונתיים (DR) או הגבלה קלורית (CR). Caenorhabditis elegans נמטודות התפתחה אורגניזם מודל חשוב עבור מחקר על הביולוגיה של ההזדקנות. מניפולציות הן סביבתיים וגנטיים שימשו מודל DR ו הראו כדי להאריך את תוחלת החיים ב C. elegans. עם זאת, רבים דיווחו DR מחקרים ב C. elegans נעשו על ידי הפצת חיות בתקשורת נוזלי, בעוד רוב המחקרים הגנטיים בתחום הזדקנות נעשו על אגר מוצק רגיל צלחות פטרי. כאן אנו מציגים פרוטוקול DR באמצעות תקן NGM אגר מוצק מבוססי צלחת עם חיידקים נהרג.

Watch this Scientific Journal Video about Solid Plate-based Dietary Restriction in Caenorhabditis elegans at JoVE.com

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

כאן אנו מציגים פרוטוקול לביצוע הגבלה מוצק צלחת מבוססי תזונתיים<em&gt; ג elegans</em&gt; עם חיידקים נהרג.

Solid פלייט מבוססי הגבלת תזונתיים ב Caenorhabditis elegans</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

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Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K Views.  University of Michigan. כאן אנו מציגים פרוטוקול לביצוע הגבלה מוצק צלחת מבוססי תזונתיים ג elegans עם חיידקים נהרג.

Video: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

מדידת Caenorhabditis elegans חיים טווח על מדיה מוצק

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

בידוד במבחנה הפעלת Caenorhabditis elegans זרע

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

זמן לשגות מיקרוסקופית של העובר בשלב מוקדם Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

בסיסי<em&gt; Caenorhabditis elegans</em&gt; שיטות: סנכרון תצפית

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

הפקה וניתוח בפרוסטגלנדין<em&gt; Elegans Caenorhabditis</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

תוספת תזונתית של חומצות שומן רב בלתי רוויות ב<em&gt; Elegans Caenorhabditis</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

טיפוח<em&gt; Elegans Caenorhabditis</em&gt; ב בשלושה ממדים במעבדה

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

מדידה של קצב הצריכה של חמצן בשלם Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

כימות ירידה פרוטאוסטטית ספציפית לרקמות ב elegans Caenorhabditis

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

אוטומציה של כימות מצטבר ב - Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...