אנו מודים למעבדת O&#39;Bryan בUIC להערכה ניסויית ביקורתית ודיון. עבודה זו נתמכה על ידי איגוד הלב האמריקאי גרנט 11SDG5230003 למרכז GKC והלאומי לקידום מדע Translational - מרכז UIC למדעים קליניים translational גרנט UL1TR000050.

1. Transfection הסלולרי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פלייט תאי היום לפני transfection, תאי ציפוי פחות עבור immunofluorescence. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לimmunofluorescence: בצלחת, מכסה מחליק זכוכית מעיל 6 גם (1 לכל טוב) עם 0.02% ג&#39;לטין על 37 מעלות צלזיוס במשך לפחות 4 שעות, לשטוף פעם עם מדיום, וגם לכל אחד, צלחת 1 x 10 5 HEK 293T 2 תאים במדיום גידול מ&quot;ל מלא [התקשורת השונה Dulbecco של הנשר (DMEM) בתוספת 10% FBS]. </li><li style=";text-align:right;direction:rtl"> למנתח כתם זרימת cytometric והמערבי: צלחת 1.2 x 10 5 תאי HEK 293T / גם במדיום גידול 2 מ&quot;ל שלם בצלחת 6 היטב (0.3 10 293T תאי x 5 HEK ב0.5 מדיום גידול שלם מ&quot;ל לכל טוב בצלחת 24 גם ). </li></ol></li><li style=";text-align:right;direction:rtl"> הכן את ה-DNA לtransfection פי הפרוטוקול של הייצור: Effectene (Qiagen) משמש לimmunofluorescence. Transit-LT1 (Mirus) משמש למנתח כתם זרימת cytometric והמערבי. כמות ה-DNA משמש היא מפורטת להלן. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><tד&gt; </td><td> צלחת 24 גם (נ&quot;ג) </td><td> צלחת 6 היטב (נ&quot;ג) </td></tr><tr><td> CFP וקטור </td><td> 10 </td><td> 50 </td></tr><tr><td> VN N-AKAP-LBC </td><td> 200 </td><td> 1000 </td></tr><tr><td> N-VC PDE4D3-FL (באורך מלא PDE4D3) </td><td> 2.5, 5, 10, 20, 40, 80 </td><td> 100 </td></tr><tr><td> VC-N-PDE4D3 UCR1 (שבר PDE4D3 N-מסוף המכיל תחום UCR1) </td><td></td><td> 100 </td></tr><tr><td> VC-N-PDE4D3 UCR2 + חתול (שבר בטרמינל C-PDE4D3 המכיל את תחומי UCR2 וקטליטי) </td><td></td><td> 100 </td></tr></tbody></table> Transfection באמצעות Effectene (6 היטב): <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף סכום המצוין של פלסמיד דנ&quot;א + 4 משפר μl עד 100 μl החיץ EC, לערבב, ודגירה במשך 5 דקות בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> הוסף 5 Effectene μl, לערבב, ודגירה במשך 10 דקות בשעהטמפרטורת חדר. הוסף טיפה חכמה לתאים. </li></ol> Transfection באמצעות מעבר-LT1 (6-well/24-well): <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף סכום המצוין של ה-DNA פלסמיד ל250 μl/50 μl של מדיום סרום ללא Opti-ממ אני בצינור סטרילי. </li><li style=";text-align:right;direction:rtl"> הוסף מגיב Transit-LT1 לתערובת ה-DNA בדילול מלא ופיפטה בעדינות לתערובת. (Transit-LT1 מגיב: יחס ה-DNA הוא 3 μl של-LT1 מעבר למגיב 1 מיקרוגרם של ה-DNA). </li><li style=";text-align:right;direction:rtl"> דגירה במשך 30 דקות בטמפרטורת חדר ולהוסיף טיפה חכמה לתאים. </li></ol></li></ol> 2. הכנת תא לcytometry זרימה, כתם מערבי, וImmunofluorescence <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 24 שעות לאחר transfection, לבדוק תחת מיקרוסקופ פלואורסצנטי epifluorescence לפני קצירת תאים. </li><li style=";text-align:right;direction:rtl"> הכנת תא לניתוח תזרים cytometric: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטוף תאים עם 0.5 מ&quot;ל / 2 מ&quot;ל קר כקרח PBS עבור צלחות 24-well/6-well. </li><li style=";text-align:right;direction:rtl"> ניתוק תאים באמצעות 50 0.05% טריפסין μl μl/250. </li><li style=";text-align:right;direction:rtl"> לנטרל טריפסין עם 200 μl / בינוני 1 מיליליטר DMEM. </li><li style=";text-align:right;direction:rtl"> שיתוף250 תאי μl llect (גם בצלחת 24 גם ו6 היטב) על ידי צנטריפוגה ב XG 500 במשך 5 דקות, השתמשו בתאים הנותרים (1 מ&quot;ל) לניתוח כתם מערבי. </li><li style=";text-align:right;direction:rtl"> Resuspend תאים ב250 חיץ FACS μl [PBS + 0.5% (w / v) BSA + 2 מ&quot;מ EDTA], לאחסן על קרח לניתוח תזרים cytometric. יכולים גם להיות קבועים בתאי 1% paraformaldehyde (PBS), אם ניתוח תזרים cytometry לא יהיה מיידי. </li></ol></li><li style=";text-align:right;direction:rtl"> הכנת תא לניתוח כתם מערבי: מתבצעת במקביל להכנת תא לניתוח cytometry זרימה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטפו תאי 1x עם 2 מ&quot;ל קר כקרח PBS, lyse תאים על ידי הוספת 100 μl של חיץ תמוגה תא [10 מ&quot;מ חיץ נתרן פוספט, pH 6.95, 150 מ&quot;מ NaCl, 5 מ&quot;מ EDTA, 5 מ&quot;מ EGTA, 1% טריטון X-100] . </li><li style=";text-align:right;direction:rtl"> לאסוף lysate תא על ידי צנטריפוגה במשך 10 דקות ב 21,000 XG ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> לכמת ריכוז חלבון על ידי assay החלבון ברדפורד, טען כמויות שווה של חלבון לכל נתיב ל- SDS, ולהעביר לnitrocellulose לimmunoblotting. </li></ol&gt; </li><li style=";text-align:right;direction:rtl"> הכנת תא לimmunofluorescence. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יש לשטוף את תאי 3x עם 2 מ&quot;ל PBS, לתקן את התאים ב 1 מ&quot;ל של paraformaldehyde 3.7% (PBS) למשך 10 דקות בטמפרטורת חדר, ולאחר מכן לשטוף 3x עם 2 מ&quot;ל PBS במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> הר לכסות להחליק על שקופיות זכוכית, באמצעות הרכבה בינונית. לאטום את קצות coverslip באמצעות לק במידת צורך. </li><li style=";text-align:right;direction:rtl"> אחסן את השקופיות על 4 מעלות צלזיוס לפני הבדיקה. </li></ol></li></ol> 3. Cytometry זרימה ומיקרוסקופיה immunofluorescence <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> cytometry הזרימה מתבצע באמצעות Beckman Coulter ציאן ADP, המצויד ב488 ננומטר, 405 ננומטר, ו642 לייזרים של מצב מוצק ננומטר. תוכנת פסגה משמשת לרכישה וניתוח. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לכייל cytometer זרימה באמצעות חרוזים סטנדרטיים. </li><li style=";text-align:right;direction:rtl"> פיזור עלילה קדימה (FSC) / פיזור צידה (SSC) בקנה מידה ליניארי לאוכלוסיית תא חי gating. </li><li style=";text-align:right;direction:rtl"> רוחב הפולס FSC / מתח מגרש לאוכלוסיית תא חי שאינם מצטבר gating. </li><li style=";text-align:right;direction:rtl"> עלילת count/FL6 (CFP fluorescencE, 405 ננומטר) בקנה מידת יומן לתאי חיים שאינם מצטברים gating CFP-חיוביים. </li><li style=";text-align:right;direction:rtl"> העלילה count/FL1 (YFP הקרינה, 488 ננומטר) בקנה מידה לעוצמת יומן BiFC. </li><li style=";text-align:right;direction:rtl"> הפעל דגימות שליליות YFP-CFP כפול, להתאים FSC, SSC, FL1 וFL6 צינור מכפיל תמונה (PMT) מתח כדי לקבוע את הסף לכל אות. </li><li style=";text-align:right;direction:rtl"> הפעלה אחת חיובי (גם YFP + +) וCFP פיצוי לא מחובר לעתיד הדגימות </li><li style=";text-align:right;direction:rtl"> לרכוש לפחות 10,000 אירועים מגודרים (CFP + תאי חיים שאינם מצטברים) לניתוח. </li></ol></li><li style=";text-align:right;direction:rtl"> מיקרוסקופיה immunofluorescence מתבצעת באמצעות מיקרוסקופ confocal 510 Zeiss LSM. הערה: חשוב לשמור על כל ההגדרות (כמו זום, חריר, עלייה בגלאי, מגבר קיזוז, גודל מסגרת, מהירות סריקה, סריקת ממוצע, וכוח לייזר) עקבי בין דוגמאות להשוואה נכונה של כל הנתונים. </li></ol> 4. ניתוח נתונים (שבוצע באמצעות תוכנות פסגה) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להגדיר את פרוטוקול הניתוח הדומה לזה לacquisitיון עם gating התקין: שער 1 בעלילת FSC / SSC נקודה לתאים חיים. שער 2 ברוחב FSC / דופק של שער 1 לתאי חיים שאינם מצטברים. שער 3 בספירה / CFP של שער 3 לCFP אינו מצטבר + תאי חיים. </li><li style=";text-align:right;direction:rtl"> לפצות YFP ואות CFP באמצעות YFP + + וCFP תאים חיוביים יחידים. </li><li style=";text-align:right;direction:rtl"> Re-קובעים קווים חתוכים לתאי CFP / YFP-חיובי. </li><li style=";text-align:right;direction:rtl"> יצוא YFP מתכוון עוצמת הקרינה (MFI) של תאי CFP + ל-Excel לנתונים ההתוויה. </li><li style=";text-align:right;direction:rtl"> לנרמל YFP MFI לרמות ביטוי של AKAP-LBC, PDE4D3, וטעינת שליטת α-טובולין, כפי שנקבע על ידי כתם מערבי. </li></ol>

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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

BiFC הוא שיטה פשוטה ורגישה ללמוד אינטראקציה בין חלבונים. שיטה זו אינה יכולה לשמש כדי לזהות חלבונים אינטראקציות חדשות, לעומת זאת, זה נוח במיוחד כדי לאשר את חלבונים האינטראקציה בתוך תאים וללמוד מאפיינים תפקודיים; כגון לוקליזציה subcellular של קומפלקסי חלבונים, מיפוי של אתרי א...

650,000 אינטראקציות בין חלבונים מוערכות להתקיים בinteractome האנושי, ממלאים תפקידים קריטיים בשמירה על 1,2 פונקציות רגילה של תא. חוץ משיתוף immunoprecipitation (שיתוף ה-IP), את תקן הזהב ללמוד אינטראקציה בין חלבונים מlysate התא, כמה מבחני שלמת בר חלבון (PCA) פותחו כדי לשפר את הרגישות באיתור חלבונים אינטראקציה בתוך תאים 3. טכניקות כוללות העברת פורסטר תהודה אנרגיה (סריג), תהודת העברת אנרגיה פליטת אור (ברט), ושלמת הקרינה bimolecular (BiFC) 4,5. BiFC מבוסס על עמותת הנחייתם של שני שברי חלבון פלואורסצנטי (כאן אנו משתמשים ונוס; גרסת YFP), כי הם כל התמזגו לשותף חלבון אינטראקציה פוטנציאלי (בדוגמה זו אנו משתמשים AKAP-LBC וPDE4D3). אינטראקציה של שני החלבונים של עניין בתוך תוצאות תאים בקרינה פונקציונלית, אשר ניתן דמיינו ידי Fמיקרוסקופיה luorescence או באמצעות תאי חיים או קבועים 6,7,8. בהשוואה לPCAs אחר, BiFC הוא רגיש ופחות מאתגר מבחינה טכנית, עם פוטנציאל ללמוד לוקליזציה הסלולר בתאים חיים. חסרון עיקרי של שיטה זו הוא שיצר עם זאת פעם אחת, לא יכול להיות הפוך מורכב חלבון פלואורסצנטי. לכן זה לא שיטה טובה ללמוד אינטראקציה בין חלבונים דינמיים. במאמר זה, אנו משתמשים BiFC למפות את האתרים של האינטראקציה עם PDE4D3 AKAP-LBC על ידי ניטור את עוצמות הקרינה של ונוס. בהשוואה לניתוח מסורתי באמצעות מיקרוסקופ פלואורסצנטי, שהוא גוזל זמן והעבודה אינטנסיבי (אם לא מתבצע באמצעות מכונות אוטומטיות תפוקה גבוהה), cytometry הזרימה מספק ניתוח הכמותי פשוט של אלפי תאים באוכלוסייה הטרוגנית על פני זמן קצר 9, 10. כאן, על ידי ביצוע השוואת Side-by-צד של זרימת ניתוח ושיתוף IP מסורתי cytometric-BiFC, אנו מדגימים כי שתיים מ &#39;ethods לספק נתונים דומים, עם זאת, ניתוח תזרים cytometric BiFC הוא פחות זמן רב ומשתמש בפחות חומר שעשויה להיות שימושיות יותר כאשר תאים של עניין מוגבלים.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle's media (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin (pen/strep), 100x</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Flag antibody</td>
			<td>Sigma</td>
			<td>M2, F1804</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Covance</td>
			<td>16B12, MMS-101R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;-tubulin</td>
			<td>Sigma</td>
			<td>DM1A, T9026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Clontech</td>
			<td>632569</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6-well tissue culture treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maxiprep plasmid purification kit, high speed</td>
			<td>Qiagen</td>
			<td>12663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-buffered saline (DPBS), sterile 1x</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05% (w/v)</td>
			<td>Gibco</td>
			<td>25300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tubes for FACS staining</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>S74337MF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P-36931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superfrost plus Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fisherfinest premium cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>CO2 air-jacketed Incubator</td>
			<td>NuAIR DH autoflow</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope LSM510 META</td>
			<td>Carl Zeiss, Inc</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrophoresis and transfer unit</td>
			<td>Biorad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cyan ADP Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction

בין שיטות ללימוד אינטראקציה בין חלבונים בתוך תאים, שלמת הקרינה bimolecular (BiFC) היא יחסית פשוטה ורגישה. BiFC מבוסס על הייצור של הקרינה באמצעות שני שברים שאינם ניאון של חלבון פלואורסצנטי (ונוס, גרסת חלבון פלואורסצנטי צהובה, משמש כאן). ברי ונוס ללא ניאון (VN והון סיכון) הם התמזגו לשני חלבוני אינטראקציה (במקרה זה, AKAP-LBC וPDE4D3), מניב הקרינה עקב אינטראקציה VN-AKAP-LBC-VC-PDE4D3 וההיווצרות של חלבון פלואורסצנטי פונקציונלי בתוך תאים. BiFC מספק מידע על לוקליזציה subcellular של קומפלקסי חלבונים ואת כוחו של אינטראקציות חלבון המבוססות על עוצמת הקרינה. עם זאת, ניתוח BiFC באמצעות מיקרוסקופ כדי לכמת את כוחו של האינטראקציה בין חלבונים הוא זמן רב ומעט סובייקטיבית בשל ההטרוגניות בביטוי חלבון ואינטראקציה. על ידי צימוד זרימת cytometricניתוח עם מתודולוגיה BiFC, אות ניאון BiFC האינטראקציה בין חלבונים ניתן למדוד במדויק לכמות גדולה של תאים בזמן קצר. הנה, אנחנו מדגימים יישום של מתודולוגיה זו כדי למפות אזורים בPDE4D3 הנדרשים לאינטראקציה עם AKAP-LBC. המתודולוגיה תפוקה גבוהה זה יכול להיות מיושם על גורמים המווסתים את הקרנת האינטראקציה בין חלבונים.

<html> PDE4D3 מבנים (איור 1) AKAP-LBC והיו התמזגו לVN ושברי הון סיכון, בהתאמה. אינטראקציה של PDE4D3 תוצאות תפקודית YFP הקרינה (איור 1 א) AKAP-LBC ו. כאן אנו משתמשים בשיטת BiFC לאתרי AKAP-LBC מחייבים המפה בPDE4D3. אזור נשמרים במעלה הזרם 1 (UCR1), באזור נשמרים במעלה הזרם 2 (UCR2) ואזור קטליטי...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

ניתוח תזרים cytometric של שלמת הקרינה bimolecular מספק שיטה כמותית תפוקה גבוהה ללמוד אינטראקציה בין חלבונים. מתודולוגיה זו יכולה להיות מיושמת על אתרי קישור ומיפוי חלבון לסינון גורמים המווסתים את האינטראקציה בין חלבונים.

ניתוח תזרים cytometric של שלמת הקרינה bimolecular: שיטה כמותית תפוקה גבוהה לחקר אינטראקציה בין חלבונים

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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Research

JoVE Journal

Biology

18.3K Views.  University of Illinois at Chicago. ניתוח תזרים cytometric של שלמת הקרינה bimolecular מספק שיטה כמותית תפוקה גבוהה ללמוד אינטראקציה בין חלבונים. מתודולוגיה זו יכולה להיות מיושמת על אתרי קישור ומיפוי חלבון לסינון גורמים המווסתים את האינטראקציה בין חלבונים.

Video: Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

עצב Astrocyte שותף תרבות Assay לניתוח תכולה גבוהה של neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

שיטת תפוקה גבוהה ברחבי העולם כדי ללמוד את המורפולוגיה של אברון cerevisiae ס

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolecular השלמה פלואורסצנטי (BiFC) Assay עבור אינטראקציה בין חלבונים בתאים בצל באמצעות אקדח הליוס ג&#39;ין

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

השלמה Fluorescence bimolecular

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

איתור של אינטראקציות חלבון הצמח באמצעות bimolecular תואם Gateway הקרינה השלמה (BiFC) מערכת

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

שלב נוזלי זיקה לכידת Assay באמצעות חרוזים מגנטיים ללמוד חלבונים אינטראקציה: דוגמה poliovirus-Nanobody

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

ניתוח כמותי של Autophagy באמצעות מיקרוסקופ פלואורסצנטי 3D מתקדם

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Assay עקידת התפוקה גבוה MHC II לניתוח כמותי של פפטיד אפיטופים

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

חלבונים אינטראקציות דמיינו ידי bimolecular הקרינה complementation בProtoplasts הטבק ועלים

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

ניתוח תפוקה גבוהה תמונה של spheroids גידול: יישום תוכנה ידידותי למשתמש כדי למדוד את גודל של spheroids באופן אוטומטי ומדויק

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...