The authors thank Dr. Michael N. Sack (National Heart, Lung, and Blood Institute) for support and discussion, Ms. Ann Kim for optimizing the B cell isolation protocol, Dr. Joseph Brzostowski for his help with microscopy, and Ms. Mirna Pe&#241;a for maintaining the animals used. This study was supported by the Intramural Research Programs of the National Institutes of Health, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute.

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The authors declare that they have no competing financial interests. Open Access fees for this article were provided by Agilent Technologies.

הפרוטוקול שפתחנו מותר בידוד יעיל של תת הלימפוציטים טהורים קיימא, אשר לאחר מכן שמשו בניתוח תאיים שטף בריכוזים שונים כדי להעריך את ההבדלים הגליקוליזה וביצועי נשימה המיטוכונדריאלי. פרוטוקול זה הוא תוכנן במיוחד עבור לימפוציטים ומספקים מענה שיקולים המיוחדים הקשורים סוגי תאים אלה, כגון פעילות חילוף חומרים בסיסית נמוכה, שבריריות, בתדירות נמוכה, וחוסר היכולת שלהם לדבוק צלחות assay. לכן, לעומת פרוטוקולים שפורסמו בעבר 11,12, השיטה שלנו מציע מדריך נוח יותר אופטימיזציה טובה יותר עבור חוקרים העובדים בתחום האימונולוגיה. ישנם מספר שלבים קריטיים בפרוטוקול זה, כולל פעולת כניסת אוכלוסיות תאים טהורות קיימא, ציפוי התאים בנקודת מפגש אופטימלי, סטנדרטיזציה של מדידות assay שטף תאי על ריכוז החלבון בכל טוב. האנימספר nitial התאים המצופים הן יכל להיות דמיינו ידי מיקרוסקופ אור וגם לכמת באמצעות מדידות ריכוז חלבון. המתאם הליניארי בין מספר תא ריכוז חלבון אשר כי ריכוז החלבון אכן יכול לשמש כדרך לתקנן את השפעת השינויים מספרים סלולריים בין בארות שונות. על ידי סטנדרטיזציה את תוצאות ECAR ו- OCR אל ריכוזי החלבון, הראינו כי, למרות מפגש תא תחתון, מספר המצומצם ככל 2.5 x 10 5 תאים / גם יכולים לשמש ברוב המבחנים מבלי להתפשר על איכות הנתונים. עם זאת, הגבול התחתון של תאים שיכולים לשמש נעים בין סוגי תאי מבחנים. לדוגמה, בעוד כמה כמו 1.25 x 10 5 תאים יכול לשמש למדידות B תא OCR, אפילו 2.5 x 10 5 תאים / גם לא היו מספיק כדי להעריך את הביצועים glycolytic מאותו סוג תא. לכן, כל עוד מספר הסלולרי אינו גורם מגביל, ציפוי ביותר מ90% מפגש, מתוכם כ מקביל 5 x 10 5 לימפוציטים / היטב, עדיף. אופטימיזציה נוספת ייתכן שתידרש כאשר תאים בגדלים-כגון שונים כמו מופעל בעבר-הם לימפוציטים בשימוש הבדיל באופן חלקי. בנוסף, בעת שימוש לימפוציטים עיקרי תרבותי בעבר, שאולי יש וריאצית כדאי תא בין תנאי טיפול שונים, אשר תקטין את האמינות של ריכוז חלבון כמדד את המספר הסלולרי מאז מת או תאים מתים יכולים גם לתרום רמות החלבון הנמדדות . במקרים כאלה, זה עשוי להיות מועיל כדי למיין תאי חיים על ידי זרימת cytometry לפני ביצוע מבחני שטף התאי. המבחנים שלנו באמצעות אוכלוסיות לימפוציטים מבודדות טרי מאוד קיימא, השגנו נתונים תפקודיים אמינים עבור שתי מדידות OCR ו ECAR. בעוד כל סוגי התאים התנהגו באופן דומה במבחן הלחץ המיטוכונדריה, הבדלים בולטים בין סוגי התאים היושנצפה מבחן הקיצון הגליקוליזה. למשל, את ביצועי glycolytic של תאי T הנאיביים היו נמוכים בהשוואה splenocytes או תאי B, וזה לא השתנה עם התוספת של oligomycin. תצפית זו עולה בקנה אחד עם מחקרים שפורסמו בעבר 7,30, המאשר את תוקפו של הפרוטוקול שלנו. לסיכום, השיטה שלנו מציעה דרך יעילה ונוחה של בדיקת הפעילות המטבולית של לימפוציטים באמצעות מנתח שטף תאי, וזה יכול להיות שימושי במגוון רחב של מחקרים אימונולוגית לחקור שינויי מטבוליים בתאי מערכת חיסוניים באקטיבציה, התמיינות תאים או בשל פנוטיפים המחלה כגון ממאירות זיהום, אוטואימוניות המטולוגיות.

התגובה החיסונית נגד אנטיגנים איזון הדוק מוסדר בין ההפעלה החיסונית דיכוי המערכת החיסונית. הפעלה חיסונית כונני התפשטות נדידת תאים מהירים, כמו גם ייצור ציטוקינים, הפרשת נוגדנים phagocytosis גדל ו בתגובה את החומר מהגוף, ואילו דיכוי מערכת חיסוני פשוט מעכב את האירועים הללו, ולכן הוא חשוב במניעת 1-6 תגובות חיסוניות מיותרות. מחקרים שנעשו לאחרונה הראו כי קיים קשר ישיר בין מצב ההפעלה של תאי מערכת החיסון ופעילות מסלולים מטבוליים שונים 7. תאי חיסון יכולים לעבור בין המדינות ההמנוחות והופעל על ידי מעבר מסלולים להפקת אנרגיה לסירוגין. יתר על כן, היא נצפתה כי סוגי תאים חיסוניים שונים עשויים להשתמש באסטרטגיות מטבוליים שונות כדי לתדלק צרכי האנרגיה המוגברות שלהם במהלך הפעלה. לדוגמא, בעוד הפעלה של לימפוציטים מסוג T מפנה תאים למצב glycolytic כמעט לחלוטין 8 </sup&gt;, לימפוציטים מסוג B מופעל להשתמש איזון של זירחון הגליקוליזה חמצוני 9,10. מחקרים אלה מצביעים על החשיבות של שבדק את ההשפעה של הפעלת תא חיסון על חילוף חומרים תאיים. בזמן אמת, מדידות סימולטני של שיעור צריכת החמצן (OCR) וקצב החמצה תאיים (ECAR), כאינדיקטורים של זרחון חמצוני הגליקוליזה, היא אסטרטגיה נפוצה להתייחס מצבי מסלולים להפקת אנרגיה 11-13. על מנת להשיג זאת, נתח שטף תאי, כגון סוסון הים XF 96, משמש באופן שגרתי. מכשיר כזה יכול להשוות שינויים במהירות OCR ו ECAR פני סוגי תאים או בתנאי גירוי שונים. עד כה, סוגי תאים שונים, כולל תאי חיסון, נחקרו באמצעות התקנים אלה. עם זאת, פרוטוקול אופטימיזציה תוכנן במיוחד עבור תאי מערכת החיסון אינו זמין. תאי מערכת החיסון, במיוחד לימפוציטים, נבדלים otסוגי התאים אותה בכמה דרכים קריטיות. לימפוציטים הם תאי שברירי כי אינם שורדים למשך זמן ארוך בתנאים vivo לשעבר 14-16. זוהי סוגיה גדולה עוד יותר כאשר הם בתרבית תקשורת צמיחה לא טובה חסרת חומרים מזינים חיוניים, כגון אלו המשמשים בניתוח שטף תאי. בניגוד מקרופאגים שורות תאים רבים, לימפוציטים אינם דבקים משטחי פלסטיק; ולכן זה קריטי לצרף אותם לצלחת הניתוח מבלי ליצור מתח. לבסוף, כמה תת-אוכלוסיות לימפוציטים עשויים להיות נדירות מאוד וקציר אותם נדרש, הכמות האופטימלית עשויה להיות מאתגר 17-19. כאן, אנו מספקים פרוטוקול אופטימיזציה כי היא פותחה במיוחד עבור לימפוציטים. שימוש splenocytes, לימפוציטים מסוג B ו לימפוציטים מסוג CD4 + T נאיבי מבודד מן הטחול הלימפה העכבר צמתים 20, אנו מציגים את המאפיינים של זירחון הגליקוליזה חמצוני במצב מנוחה שלהם בידולצפיפות תא t. נתונים היו מנורמלים להסביר את הבדלי המספרים הסלולריים הראשוניים עבור כל טוב על ידי מדידת ריכוזי חלבון lysate תא assay סוף, שהיו ביחס ישר המספרים הסלולריים. הפרוטוקול שלנו לא רק מספק הנחיות הבידוד המהיר של לימפוציטים מעשי עבור מבחני שטף תאיים, אבל זה גם מאפשר לעבודה בריכוזי תא הכי מוצלחים מבלי להתפשר על איכות הנתונים.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PBS (pH 7.2)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">20012-050</td>
			<td style="width:283px;">To make MACS buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bovine Serum Albumin BSA</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">A3803</td>
			<td style="width:283px;">To make MACS buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">0.5M EDTA, pH 8</td>
			<td style="width:139px;">Quality Biological</td>
			<td>10128-446</td>
			<td style="width:283px;">To make MACS buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">autoMACS rinsing solution</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-091-222</td>
			<td style="width:283px;">Instead of PBS + EDTA to make MS buffer. Also used in autoMACS Pro Separator.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">RPMI-1640</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">11875-093</td>
			<td style="width:283px;">Contains phenol red and L-glutamine</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">10437-028</td>
			<td style="width:283px;">For heat-inactivation, thaw frozen stock bottle in a 37 &#176;C water bath and then inactivate at 56 &#176;C for 30 minutes. Aliquot heat-inactivated serum for storage (e.g., in 50 mL conical tubes) and freeze at -20 &#176;C until needed.&#160;</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:216px;">Penicillin-Streptomycin (Pen Strep)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">15140-122</td>
			<td style="width:283px;">Combine Pen Strep with L-Glut 1:1 (if making 500mL media, make a total of 12 mL Pen Strep/L-Glut); keep aliquots at -20 &#176;C until ready to make media.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">L-Glutamine 200mM (L-Glut)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">25030-081</td>
			<td style="width:283px;">Component of RF10 and stress test media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium pyruvate 100mM</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">11360-070</td>
			<td style="width:283px;">Component of RF10 and stress test media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">HEPES 1M</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">15630-080</td>
			<td style="width:283px;">Component of RF10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">11140-050</td>
			<td style="width:283px;">Component of RF10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2-Mercaptoethanol (55 mM)</td>
			<td style="width:139px;">Gibco-ThermoFisher</td>
			<td style="width:104px;">21985-023</td>
			<td style="width:283px;">Component of RF10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Falcon 70 &#956;m cell strainer</td>
			<td style="width:139px;">Falcon-Fischer Scientific</td>
			<td style="width:104px;">87712</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Monoject 3 mL Syringe, Luer-Lock Tip</td>
			<td style="width:139px;">Covidien</td>
			<td style="width:104px;">8881513934</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Falcon 15mL Conical Centrifuge Tubes&#160;</td>
			<td style="width:139px;">Falcon-Fischer Scientific</td>
			<td style="width:104px;">14-959-53A</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Falcon 50mL Conical Centrifuge Tubes&#160;</td>
			<td style="width:139px;">Falcon-Fischer Scientific</td>
			<td style="width:104px;">14-432-22</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:104px;">10-548E</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">CellTrics 30 &#956;m cell filter, sterile, single-packed</td>
			<td style="width:139px;">Partec CellTrics</td>
			<td>04-004-2326</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">B Cell Isolation Kit, mouse</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-090-862</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Na&#239;ve CD4+ T cell isolation kit, mouse</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-104-453</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">LS Column</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td style="width:283px;">Used in cell isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MidiMACS separator</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td style="width:283px;">Magnet for separation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MACS MultiStand</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td style="width:283px;">Holder for magnets</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">autoMACS Rinsing Solution</td>
			<td style="width:139px;"></td>
			<td>130-091-222</td>
			<td style="width:283px;">Rinsing solution for autoMACS Pro Separator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">autoMACS Pro Separator Instrument</td>
			<td style="width:139px;">Miltenyi Biotec</td>
			<td style="width:104px;">N/A</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2-Deoxy-D-glucose (2-DG)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td>D8375-5G</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Cell-Tak</td>
			<td style="width:139px;">Corning</td>
			<td style="width:104px;">354240</td>
			<td style="width:283px;">Cell adhesive. Take care to note concentration, as each lot is different (package is 1 mg).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Oligomycin</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">75351</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2,4 Dinotrophenol (2,4-DNP)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">D198501</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Antimycin A</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">A8674</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Rotenone</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">R8875</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glucose</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">G8270</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Halt Protease Inhibitor Cocktail</td>
			<td style="width:139px;">ThermoFisher Scientific</td>
			<td style="width:104px;">78429</td>
			<td style="width:283px;">Supplied as 100X cocktail, combine with RIPA to form 1X solution for lysis.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">RIPA Buffer</td>
			<td style="width:139px;">Boston BioProducts</td>
			<td style="width:104px;">BP-115</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:139px;">ThermoFisher Scientific</td>
			<td style="width:104px;">23225</td>
			<td style="width:283px;">Follow manufacturer&#39;s instructions</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">Seahorse XFe96 FluxPak</td>
			<td style="width:139px;">Seahorse Bioscience</td>
			<td style="width:104px;">101085-004</td>
			<td style="width:283px;">Includes assay plates, cartridges, loading guides for transferring compounds to the assay cartridge, and calibrant solution.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Seahorse XF Base Medium</td>
			<td style="width:139px;">Seahorse Bioscience</td>
			<td style="width:104px;">102353-100</td>
			<td style="width:283px;">Used to prepare stress test media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Seahorse XFe96 Extracellular Flux Analyzer</td>
			<td style="width:139px;">Seahorse Bioscience</td>
			<td style="width:104px;"></td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Stericup Sterile Vacuum Filter Units, 0.22 &#956;m</td>
			<td style="width:139px;">EMD Millipore-Fisher Scientific</td>
			<td style="width:104px;">SCGPU10RE</td>
			<td style="width:283px;">Used to sterile filter media.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium bicarbonate</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:104px;">487031</td>
			<td style="width:283px;">Dissolved to 0.1M and used to dilute Cell-Tak.</td>
		</tr>
	</tbody>
</table>

an optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes

לימפוציטים להגיב למגוון של גירויים באמצעות הפעלת מסלולי איתות תאיים, אשר בתורו מוביל לשגשוג הסלולר המהירה, הגירה והבחנה, וייצור ציטוקינים. כל האירועים הללו הם קשורים באופן הדוק את מצב האנרגיה של התא, ולכן לומד את המסלולים מייצרי אנרגיה עשוי לתת רמזים לגבי הפונקציונליות הכללית של תאים אלה. מנתח השטף התאי הוא מכשיר נפוץ להערכת הביצועים של הגליקוליזה ונשימה המיטוכונדריה סוגי תאים רבים. מערכת זו נעשה שימוש כדי ללמוד תאים חיסוניים כמה דיווחים שפורסמו, עדיין פרוטוקול מקיף מותאם במיוחד עבור לימפוציטים חסר. לימפוציטים הם תאים שברירי ששורדים גרועים בתנאי vivo לשעבר. לעתים קרובות תת הלימפוציטים הם נדירים, ועבודה עם מספרים סלולריים נמוכים היא בלתי נמנעת. לפיכך, אסטרטגיית הניסוי הנותן מענה לקשיים אלה נדרש. כאן, אנו מספקים פרוטוקולהמאפשר בידוד מהיר של לימפוציטים קיימא מרקמות הלימפה, ועל הניתוח של המדינות המטבולית שלהם מנתח השטף התאי. יתר על כן, אנו מספקים תוצאות ניסויים שבהם הפעילויות המטבולית של תת הלימפוציטים כמה בצפיפויות תאים שונים הושוו. מקרים אלה מצביעים כי הפרוטוקול שלנו יכול לשמש כדי להשיג תוצאות עקביות היטב סטנדרטיות גם בריכוזי תא נמוכים, וכך יכולה להיות שהיא כוללת יישומים רחבים במחקרים עתידיים התמקדות באפיון אירועים מטבוליים בתאי מערכת חיסוניים.

Watch this Scientific Journal Video about An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes at JoVE.com

פרוטוקול אופטימלי לניתוח גליקוליזה ונשימה מיטוכונדריאלית בלימפוציטים | אימונולוגיה וזיהום | וִידֵאוֹ

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

פרוטוקול אופטימיזציה כדי לנתח הגליקוליזה מיטוכונדריאלי הנשימה לימפוציטים

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

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29.5K Views.  National Institutes of Health. The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Video: An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

שיטות להעריך מתווכת בטא מוות של תאים על ידי לימפוציטים מסוג T ציטוטוקסיות

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

פרוטוקול אופטימיזציה עבור transfection יעילה של תאים דנדריטים ללא ההבשלה Cell

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

פרוטוקול פשוט ומהיר לללא אנזימי רקמות אנושיות טריות לנתק לניתוח להחדרה לימפוציטים

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

שיטה אופטימלית עבור בידוד והרחבת תאי Invariant הטבעי רוצח T מטחול העכבר

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

פרוטוקול בידוד יעיל עבור B ו- T לימפוציטים משקדי הפלטין אדם

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

הקמת הגדרת תפוקה גבוהה להקרנה מולקולות קטנות לווסת ג-di-GMP איתות<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

זרימה Virometry לנתח אנטיגני ספקטרה של virions ואת תאי שלפוחית

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

ברונכאלווולאר של ריאות Murine כדי לנתח דלקת תאים דלקתית

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Overexpression וטיהור האדם<em&gt; Cis</em&gt; פרנליטרונפרפרז ב<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...