This work was kindly funded by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and the Irwin Joffe Memorial Fellowship.

בהצהרת אתיקה: כל עבודת החיה נערכת עם הרישיון והאישור של משרד פן בריטניה. השתתפות אדם במחקר זה אושר על ידי ועדת UCL המשותפת / UCLH על האתיקה של מחקר אדם. כל המשתתפים סיפקו הסכמה מדעת. 1. הכנת ג אלביקנס <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדול דיסק קנדידה (ראה רשימת חומרים) על צלחת אגר YPD לפי הוראות היצרן. פיק מושבה ולהוסיף אותו 15 מ&quot;ל של מרק YPD. דגירה זה חממה רועד ב 30 ° C ו 200 סל&quot;ד עד שהמרק הוא מעונן (בדרך כלל על 2 ימים, או לריכוז של בערך מעל 1 x 10 9 / מ&quot;ל). </li><li style=";text-align:right;direction:rtl"> ספין למטה המדיום קנדידה מחדש להשעות אותו 50 מ&quot;ל של פוספט שנאגרו מלוחים (PBS). צנטריפוגה ב 3000 XG במשך 10 דקות. חזור על פעולה זו פעמיים. </li><li style=";text-align:right;direction:rtl"> מניחים את הצינור המכיל 50 מ&quot;ל של מדיום PBS / קנדידה באמבט מים שחומם מראש ל 60 מעלות צלזיוס, כך הצינור כולו הוא יםubmerged עבור 1 h. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לאשר כי קנדידה הם-הרג חום, פס מדגם לצלחת אגר YPD ו דגירה לילה בשעה 30 ° C. התאם את-הרג חום (HK) ריכוז קנדידה כדי 5-9 x 10 8 / מ&quot;ל, תלוי צמיחה קנדידה, ולאחסן אותו 1 aliquots מ&quot;ל ב -20 ° C במקפיא. הערה: כל ספירת התאים בפרוטוקול זה בוצעה באמצעות דלפק תא אוטומטי. </li></ol></li></ol> 2. S-1 צימוד חום-הרג (HK) קנדידה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן aliquot של carboxy-S-1 אסתר succinimidyl (50 מיקרוגרם) על ידי דילול אותו 100 μL של sulfoxide דימתיל בדרגה גבוהה (DMSO). וורטקס היטב כדי לערבב. </li><li style=";text-align:right;direction:rtl"> הכן 1 מ&quot;ל של 1 x 10 8 קנדידה HK ב 0.1 M סודיום ביקרבונט (pH 8.3) בצינור 15 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הוספת 100 μL של carboxy-S-1 טיפה אחת בכל פעם, כדי קנדידה HK תוך ערבוב על מערבולת בערך 2000 סל&quot;ד (בינוני עד מהיר). גלישת אלומיניום foil סביב הצינור ולמקם אותו על רולר בטמפרטורת החדר למשך 1 שעה. </li><li style=";text-align:right;direction:rtl"> לשטוף שלוש פעמים HK Candida- S-1 (HKC-S-1) על ידי צנטריפוגה ב 2250 XG במשך 10 דקות כל אחד. בשנתי השטיפות הראשונות, להשעות את הגלולה ב 15 מיליליטר של 0.1 M סודיום ביקרבונט (pH 8.3). לאחר לשטוף השלישי, להשעות אותו 1 מיליליטר של תמיסת מלח מאוזן (BSS) חיץ (ראה טבלה 1). </li><li style=";text-align:right;direction:rtl"> העברת HKC-S-1 ההשעיה לתוך צינורות ב 100 aliquots μL ולאחסן אותם ב -20 ° C. הערה: צינורות משתמשים עם משטח נמוך מחייב חומר, ואם אפשר, כפי HKC-S-1 חלקיקים יכולים להיצמד לקיר של צינורות צנטריפוגה נורמלים. </li><li style=";text-align:right;direction:rtl"> Opsonize HKC-S-1 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עבור נויטרופילים האדם, להוסיף 100 μL של נסיוב אדם IgG ל 100 μL של HKC-S-1 מופשר. </li><li style=";text-align:right;direction:rtl"> עבור נויטרופילים עכבר, להוסיף 50 μL של סרום עכבר רגיל 50 μL של בסרום חיסוני עכבר קנדידה (המופקים עכברי C57B6 מוזרקים עם קנדידה HK) 5 כדי 100 μLשל HKC-S-1. </li><li style=";text-align:right;direction:rtl"> מערבבים אותו על-שייקר חום ב 37 ° C ו- 1100 סל&quot;ד במשך 60-90 דקות, ולאחר מכן על רולר C 4 ° עבור 2 h. </li><li style=";text-align:right;direction:rtl"> שלוש פעמים לשטוף במאגר BSS על ידי צנטריפוגה ב 17,200 XG במשך 1 דקות כל אחד. גלולה ב 100 μL של חיץ BSS. </li></ol></li></ol> 3. בידוד של נויטרופילים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עבור נויטרופילים בדם ההיקפי האנושי, לקחת 15 מ&quot;ל של דם על ידי venipuncture מתורם בריא ולמקם אותו לתוך מזרק 20 מ&quot;ל המכיל 90 μL של 1000 פתרון נתרן הפרין IU / מ&quot;ל ​​(60 μL של הפרין / 10 מ&quot;ל של דם). </li><li style=";text-align:right;direction:rtl"> בעזרת קצה פיפטה, להזריק 1.5 מ&quot;ל של פתרון dextran 10% לתוך המזרק. הפוך אותו בעדינות 3 פעמים ולהשאיר אותו עומד זקוף על הספסל. </li><li style=";text-align:right;direction:rtl"> המתן 30-60 דקות, עד שהדם מתפרקים לשני חצאים, עם שכבת מעיל באפי העליון כי הוא בצבע קש שכבה בתחתית המכיל אריתרוציטים. </li><li style=";text-align:right;direction:rtl"> לחץ בעדינות את השכבה העליונה באמצעות מחט או להסיר אותו עם קצה פיפטה. שים את זה אניצינור נה 15 מ&quot;ל. להימנע מלקחת את השכבה האדומה. בעזרת פיפטה מ&quot;ל 5, להוסיף 3-4 מ&quot;ל של מדיום שיפוע צפיפות (ראה רשימת חומרים) לחלק התחתון של הצינור מתחת לשכבת מעיל באפי להשיג שתי שכבות נפרדות. צנטריפוגה ב 913 XG במשך 10 דקות. הערה: אם נויטרופילים עכבר באמצעות (ראו התייחסות 17 לעכבר נויטרופילים בידוד), להשתמש השעית התא כי כבר סמוקה ממח העצם במקום. </li><li style=";text-align:right;direction:rtl"> יוצקים את supernatant, ומשאיר מאחור גלולה אדומה. בעדינות מערבולת להפריע גלולה. להוסיף 7 מ&quot;ל של מזוקקים, לעקר H 2 O ל גלולה ו להפוך עבור 20 s כדי להשעות את גלולה. להוסיף 7 מ&quot;ל של סליין 2x ו להפוך כמה פעמים כדי לערבב, lysing בתאי הדם האדומים שנותרו. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה XG 300 5 דק &#39;. יוצקים את supernatant מחדש להשעות גלולה במאגר BSS כ 4 x 10 6 / מ&quot;ל. הערה: למיקרוסקופיה האופטימלי של התאים, לשמור על ריכוז השעית תא בין 1.5-6 x 10 6 </sup&gt; / מ&quot;ל. </li></ol> 4. הכנת שקופיות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> טרום לטפל צלחת מיקרוסקופיה 8 היטב (ראה רשימת חומרים) עם 200 μL של L- ליזין פולי (פתרון 0.01%) בכל טוב עבור 40-60 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הסר את L- ליזין פולי (ניתן לעשות שימוש חוזר) ולשטוף את הבארות פעמיים עם 200 μL של H 2 O. מזוקקים </li><li style=";text-align:right;direction:rtl"> הוספת 200 μL של תאי השעיה מוכנות בשלב 3.6 זה טוב. דגירה בטמפרטורת החדר למשך 30-60 דקות. </li><li style=";text-align:right;direction:rtl"> הכן aliquot של 5 (ו-6) acetoxymethyl S-1 -carboxy (S-1-AM) אסתר (50 מיקרוגרם) על ידי הוספת 100 μL של DMSO בדרגה גבוהה כדי צינור אחד. וורטקס לערבב. כאשר אינו בשימוש, ולאחסן ב -20 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> בתוך צינור קטן, להוסיף 1.7 מ&quot;ל של חיץ BSS ו- 20 μL של S-1-AM. וורטקס לערבב. </li><li style=";text-align:right;direction:rtl"> לשטוף את הבארות פעמיים עם 200 μL של חיץ BSS, ולאחר מכן להחליף את החיץ עם הפתרון S-1-AM. יש להיזהר שלא להפריע בשכבה של תאים המחוברים לתחתית הבאר ידי בעדינות pipettingלאורך הקירות של הבארות. דגירה בטמפרטורת החדר למשך דקה 25 לפחות. </li><li style=";text-align:right;direction:rtl"> לשטוף את הבארות פעמיים עם 200 μL של חיץ BSS. כדי לבחון מעכב, לפצות על &quot;תערובת מאסטר&quot; של התרופה המתאימה חיץ BSS. לשטוף את הבארות פעמי הפתרון המעכב. הערה: הקפד להשתמש באותה כמות של הרכב התרופה (למשל, DMSO או אתנול) בבקרה גם שימש מעכב. אם בוחנים את ההשפעה של כלוריד אבץ, להשתמש במאגר BSS חסר KH 2 PO 4 (את HEPES יכול חיץ הפתרון לבד), כמו אבץ משקעים עם פוספט. </li><li style=";text-align:right;direction:rtl"> Sonicate HKC-S-1 opsonized (סעיף 6.2) למשך כ 3 שניות על מיקרומטר משרעת 5. להוסיף 10 μL היטב כל אחד. דגירה את הצלחת על 37 מעלות צלזיוס למשך 15-20 דקות, כדי לאפשר phagocytosis, מה שהופך את התא מוכן להיות צלם עבור תמונות של phagocytosis. </li></ol> 5. מיקרוסקופיה confocal <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות מיקרוסקופ confocal, להתאים את אורך הגל לייזר כך thעל התאים נרגשים ב 555 ננומטר ואת הפליטה מזוהית על ידי שני ערוצים, או גלאי: 560-600 ננומטר ומעל 600 ננומטר. הערה: פרמטרים מיקרוסקופ ספציפיים יהיו שונים עבור כל מיקרוסקופ צריכים להיות מותאמת על ידי החוקר. </li><li style=";text-align:right;direction:rtl"> הצג את התאים בעזרת עדשה 63X עם שמן. השתמש בתמונת אריח-סריקה על הגדרה רציפה כדי להציג את משבצת המרכז. באמצעות עוצמת הקרינה ורווח של ערוצי גלאי, לשנות את הפוקוס ואת עוצמת הלייזר ואת הרווח של שני הערוצים כדי לייעל את התמונה. </li><li style=";text-align:right;direction:rtl"> לפצל את התמונה באמצעות הגדרות כדי להציג שני הערוצים; לבדוק שאין רוויה של עוצמת קרינה בציטופלסמה או וקואולות. רוויה מופיעה כנקודות אדומות. להפחית את עוצמת הלייזר כך שיש מספר מינימלי של נקודות אדומות, אבל התאים phagosomes הם חכמים מספיק כדי לראות בבירור. </li><li style=";text-align:right;direction:rtl"> כשתהיה מרוצה, לחץ על &quot;התחל ניסוי.&quot; תמונת סריקה 9 אריח נוצרת. שמור את התמונה כקובץ מורכב המומלץ על ידיהתוכנה המכילה תמונה מורכבת של שני הערוצים (לא JPEG או TIFF). </li></ol> 6. ניסויי כיול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בניית עקומת סטנדרט pH עם קנדידה לבד. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את החוצצים בין pH 3.0 כדי 13.0, לפי טבלה 1. </li><li style=";text-align:right;direction:rtl"> קח aliquot אחד (100 μL) של HKC-S-1. לסובב אותו על 1 דקות ב g 17,200 x. Resuspend אותו 500 μL של חיץ שהוקצו. </li><li style=";text-align:right;direction:rtl"> החל מ טווח pH הנמוך ביותר, להוסיף את ההשעיה לאחד טרום יחס טוב (משלב 4.2). מניחים את השקף על מיקרוסקופ confocal על הבמה מחוממת מוגדר 37 ° C. </li><li style=";text-align:right;direction:rtl"> רשום את הקרינה בכל דקה במשך 10 דקות. חזור עבור כל חיץ. </li></ol></li><li style=";text-align:right;direction:rtl"> עקומת סטנדרט Saponin. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבודד 1 x 10 7 נויטרופילים האנושי 5 resuspend אותם 1 מ&quot;ל של חיץ pH הנמוך ביותר. </li><li style=";text-align:right;direction:rtl"> בצע את השיטה המתוארת בסעיף 4 למדידת ppH hagosomal ב נויטרופילים. </li><li style=";text-align:right;direction:rtl"> לאחר הדגרה עם ההשעיה קנדידה עבור 20 דקות, להוסיף 0.3% saponin (15 μL של 10% במלאי לאחד טרום יחס טוב (משלב 4.2)) על הצלחת, ואז דגירה במשך 20 דקות ב 37 מעלות צלזיוס. קח תמונה כל דקה במשך 10 דקות. חזור עבור כל חיץ. </li></ol></li><li style=";text-align:right;direction:rtl"> עקומות סטנדרטיות pH Cytosolic באמצעות הגבוהה K + / nigericin טכניקה 5, 18, 19. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> איפור מאגרים שמתואר בטבלה 1, מ pH 4.0 כדי 13.0. </li><li style=";text-align:right;direction:rtl"> בצע את פעולות 4.1-4.7, עוזב נויטרופילים רק הבארות טרום טיפול - כל טוב להכיל חיץ שונה. מוסיף את השקף אל הבמה המחוממת 37 מעלות צלזיוס ולהקליט את הקרינה בכל דקה במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> הערה: זה עלול לקחת קצת זמן עבור ה- pH cytosolic לייצב עם הפתרון החיצוני, אך לאחר נקודה זו, ערך היחס S-1 הופךקָבוּעַ. מדידת HKC-S-1-התאים בכל ניסוי יכולה לשמש כביקורת לבדוק שכל מעכבים הוסיפו לא להפריע הקרינה הנפלטת S-1 או להשפיע על רמת החומציות של עור החיץ. </li></ol></li></ol> ניתוח תמונה 7. הערה: הנה, הוראות לניתוח תמונה (לכמת הקרינה) באמצעות ImageJ התוכנה החופשי מסופקת. שימוש ImageJ מומלץ. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הורד והתקן גרסה ImageJ&gt; 1.46r בהתאם להנחיות של היזם (https://imagej.nih.gov/ij/). התקן את קובץ קוד המשלים מצורף עם פרוטוקול זה נקרא &quot;מדידת Phagosomal.&quot; </li><li style=";text-align:right;direction:rtl"> פתח תמונה J ו לטעון את קובץ התמונה שנבחרה לניתוח על הכלים. כדי לשלב את שני הערוצים, להשתמש Image&gt; צבע&gt; הפוך מרוכבים. </li><li style=";text-align:right;direction:rtl"> לחץ לחיצה ימנית כדי לבחור קובץ בו כדי לאחסן תוצאות. </li><li style=";text-align:right;direction:rtl"> לחץ על כלי הקו בסרגל הכלים ולחצו לחיצה כפולה על מנת להגדיל את הרוחב ל &amp; #34; 2 &quot;. </li><li style=";text-align:right;direction:rtl"> צייר קו לרוחב של phagosome, ולאחר מכן לחץ לחיצה ימנית על &quot;pH מידה&quot;. עוצמת הממוצע של שני ערוצי נרכש, ואת היחס שלהם מחושב באופן אוטומטי על ידי קובץ קוד ImageJ. </li><li style=";text-align:right;direction:rtl"> לאחר שסיים את המדידות, לחץ לחיצה ימנית כדי לבחור &quot;לשמור את הקובץ.&quot; </li><li style=";text-align:right;direction:rtl"> כדי למדוד את שטח phagosome, השתמש בסמל הרביעי יחד בסרגל הכלים לצייר ביד חופשית מסביב לאזור. לחץ לחיצה ימנית כדי לבחור &quot;אזור מידה.&quot; הערה: התוצאות נשמרות בקובץ יומן נוצר בתיקייה הנבחרת. התוצאות עשויות להיות מעובד באמצעות גיליון אלקטרוני, R, או תוכנה מקבילה. הקשר בין יחס קרינת pH הוא כ sigmoidal, ואת ערכי היחס שנוצרו על ידי ניתוח ImageJ ניתן להמיר pH באמצעות פונקצית סיגמואיד כללית או לוגיסטית או על ידי ליניארי או אינטרפולציה שגם מעוקב. </li><li style=";text-align:right;direction:rtl"> כדי למדוד הציטופלסמה, למתוח קו על פני הציטופלסמה לבין לחץ לחיצה ימנית על &quot;pH מידה&quot;. </li></ol>

<ol>
	<li>Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidases+as+electrochemical+generators+to+produce+ion+fluxes+and+turgor+in+fungi,+plants+and+humans.">NADPH oxidases as electrochemical generators to produce ion fluxes and turgor in fungi, plants and humans.</a> Open Biol. 6 (5), 515-517 (2016).</li><li>Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+neutrophils+kill+microbes.">How neutrophils kill microbes.</a> Annu Rev Immunol. 23, 197-223 (2005).</li><li>Cross, A. R., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NADPH+oxidase+of+professional+phagocytes+-+Prototype+of+the+NOX+electron+transport+chain+systems.">The NADPH oxidase of professional phagocytes - Prototype of the NOX electron transport chain systems.</a> BBA Bioenergetics. 1657 (1), 1-22 (2004).</li><li>Demaurex, N., El Chemaly, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+roles+of+voltage-gated+proton+channels+in+leukocytes.">Physiological roles of voltage-gated proton channels in leukocytes.</a> J Physiol. 588 (23), 4659-4665 (2010).</li><li>Levine, A. P., Duchen, M. R., de Villiers, S., Rich, P. R., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alkalinity+of+neutrophil+phagocytic+vacuoles+is+modulated+by+HVCN1+and+has+consequences+for+myeloperoxidase+activity.">Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.</a> PLoS One. 10 (4), 0125906 (2015).</li><li>Reeves, E. P., Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+activity+of+neutrophils+is+mediated+through+activation+of+proteases+by+K++flux.">Killing activity of neutrophils is mediated through activation of proteases by K+ flux.</a> Nature. 416 (6878), 291-297 (2002).</li><li>Menegazzi, R., Busetto, S., Dri, P., Cramer, R., Patriarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+ion+efflux+regulates+adherence,+spreading,+and+respiratory+burst+of+neutrophils+stimulated+by+tumor+necrosis+factor-alpha+(TNF)+on+biologic+surfaces.">Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils stimulated by tumor necrosis factor-alpha (TNF) on biologic surfaces.</a> J Cell Biol. 135 (2), 511-522 (1996).</li><li>Ohkuma, S., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+measurement+of+the+intralysosomal+pH+in+living+cells+and+the+perturbation+of+pH+by+various+agents.">Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.</a> Cell Biol. 75 (7), 3327-3331 (1978).</li><li>Jankowski, A., Scott, C. C., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+phagosomal+pH+in+neutrophils.">Determinants of the phagosomal pH in neutrophils.</a> J Biol Chem. 277 (8), 6059-6066 (2002).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Indicators+of+Ion+Concentrations.">Fluorescent Indicators of Ion Concentrations.</a> Methods in Cell Biol. 30, 127-156 (1989).</li><li>Lavis, L. D., Rutkoski, T. J., Raines, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+pK+a+of+Fluorescein+to+Optimize+Binding+Assays.">Tuning the pK a of Fluorescein to Optimize Binding Assays.</a> Anal Chem. 79 (17), 6775-6782 (2007).</li><li>Hurst, J. K., Albrich, J. M., Green, T. R., Rosen, H., Klebanoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase-dependent+fluorescein+chlorination+by+stimulated+neutrophils.">Myeloperoxidase-dependent fluorescein chlorination by stimulated neutrophils.</a> J Biol Chem. 259 (8), 4812-4821 (1984).</li><li>Nunes, P., Guido, D., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Phagosome+pH+by+Ratiometric+Fluorescence+Microscopy.">Measuring Phagosome pH by Ratiometric Fluorescence Microscopy.</a> J Vis Exp. (106), e53402 (2015).</li><li>Canton, J., Khezri, R., Glogauer, M., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+phagosome+pH+regulation+and+maturation+in+human+M1+and+M2+macrophages.">Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages.</a> Mol Biol Cell. 25 (21), 3330-3341 (2014).</li><li>Schlafer, S., Dige, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+Imaging+of+Extracellular+pH+in+Dental+Biofilms.">Ratiometric Imaging of Extracellular pH in Dental Biofilms.</a> J Vis Exp. (109), e53622 (2016).</li><li>Buckler, K. J., Vaughan-Jones, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+new+pH-sensitive+fluoroprobe+(carboxy-SNARF-1)+for+intracellular+pH+measurement+in+small,+isolated+cells.">Application of a new pH-sensitive fluoroprobe (carboxy-SNARF-1) for intracellular pH measurement in small, isolated cells.</a> Pfl &#252;gers Archiv EJP. 417 (2), 234-239 (1990).</li><li>El Chemaly, A., Nunes, P., Jimaja, W., Castelbou, C., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hv1+proton+channels+differentially+regulate+the+pH+of+neutrophil+and+macrophage+phagosomes+by+sustaining+the+production+of+phagosomal+ROS+that+inhibit+the+delivery+of+vacuolar+ATPases.">Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases.</a> J Leuko Biol. 95 (5), 827-839 (2014).</li><li>Morgan, D., Capasso, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+proton+channels+maintain+pH+in+human+neutrophils+during+phagocytosis.">Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis.</a> Proc Natl Acad Sci USA. 106 (42), 18022-18027 (2009).</li><li>Thomas, J. A., Buchsbaum, R. N., Zimniak, A., Racker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH+measurements+in+Ehrlich+ascites+tumor+cells+utilizing+spectroscopic+probes+generated+in+situ.">Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.</a> Biochemistry. 18 (11), 2210-2218 (1979).</li><li>Levine, A. P., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NADPH+Oxidase+and+Microbial+Killing+by+Neutrophils,+With+a+Particular+Emphasis+on+the+Proposed+Antimicrobial+Role+of+Myeloperoxidase+within+the+Phagocytic+Vacuole.">The NADPH Oxidase and Microbial Killing by Neutrophils, With a Particular Emphasis on the Proposed Antimicrobial Role of Myeloperoxidase within the Phagocytic Vacuole.</a> Microbiol Spectr. 4 (4), (2016).</li><li>Segal, A. W., Geisow, M., Garcia, R., Harper, A., Miller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+burst+of+phagocytic+cells+is+associated+with+a+rise+in+vacuolar+pH.">The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.</a> Nature. 290 (5805), 406-409 (1981).</li><li>Hunter, R. C., Beveridge, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+pH-sensitive+fluoroprobe+(C-SNARF-4)+for+pH+microenvironment+analysis+in+Pseudomonas+aeruginosa+biofilms.">Application of a pH-sensitive fluoroprobe (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms.</a> Appl Environ Microbiol. 71 (5), 2501-2510 (2005).</li><li>Van Erp, P. E. J., Jansen, M. J. J. M., De Jongh, G. J., Boezeman, J. B. M., Schalkwijk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+measurement+of+intracellular+pH+in+cultured+human+keratinocytes+using+carboxy-SNARF-1+and+flow+cytometry.">Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry.</a> Cytometry. 12 (2), 127-132 (1991).</li><li>Minamikawa, T., Takamatsu, T., Kashima, S., Fushiki, S., Fujita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+calcium+imaging+with+ultraviolet+laser-+scanning+microscopy+and+indo-1.">Confocal calcium imaging with ultraviolet laser- scanning microscopy and indo-1.</a> Micron. 24 (6), 551-556 (1993).</li><li>Takahashi, A., Camacho, P., Lechleiter, J. D., Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Intracellular+Calcium.">Measurement of Intracellular Calcium.</a> Physiol Rev. 79 (4), (1999).</li><li>Hillson, E. J., Dewitt, S., Hallett, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+methods+for+the+measurement+and+manipulation+of+cytosolic+free+calcium+in+neutrophils.">Optical methods for the measurement and manipulation of cytosolic free calcium in neutrophils.</a> Methods Mol Biol. 412, 125-137 (2007).</li><li>Bernardo, J., Long, H. J., Simons, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+cytoplasmic+and+phagosomal+consequences+of+human+neutrophil+exposure+to+Staphylococcus+epidermidis.">Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis.</a> Cytometry Part A. 77 (3), 243-252 (2010).</li><li>Riazanski, V., Gabdoulkhakova, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC6+channel+translocation+into+phagosomal+membrane+augments+phagosomal+function.">TRPC6 channel translocation into phagosomal membrane augments phagosomal function.</a> Proc Natl Acad Sci USA. 112 (47), 6486-6495 (2015).</li><li>Mart&#237;nez-Zaguil&#225;n, R., Mart&#237;nez, G. M., Lattanzio, F., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+intracellular+pH+and+Ca2++using+the+fluorescence+of+SNARF-1+and+fura-2.">Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2.</a> Am J Physiol Cell Physiol. 260 (2), (1991).</li></ol>

The authors declare that they have no competing financial interests.

Once the appropriate reagents, microscope settings, and calibration experiments are set up, this method is relatively simple to perform. The critical steps include: labeling the Candida with S-1 to ensure that there is no overloading of the dye, calibrating, and analyzing the image.
S-1 is a reagent suited to more alkaline pH environments, which is particularly important for neutrophils21 but limits its use in certain cell types. For more acidic environments, such as macrophage phagosomes, SNARF-4, or S-4, is more suitable because of its lower pKa22. Moreover, for more accurate cytoplasmic readings, it is better to use S-4, as the standard curve for S-1 shows that fluorescence ratios begin to plateau below pH 6 (Figure 3). Other dyes, such as 2&#39;,7&#39;-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein (BCECF) or pHrodo Red may also be more suitable in a context that is expected to be acidic. Yet the cytoplasm staining is still necessary for correct identification of the phagosomes containing Candida.
An important feature of a phagosomal pH indicator is that it is not irreversibly altered by the reactive phagosomal environment. S-1 seems to be resistant to the neutrophil milieu. This is shown by Levine et al.5 (see Supplementary Video 4 of reference5) which demonstrate the phagocytosis and subsequent release of an S-1-labeled Candida particle by an Hvcn1-/- neutrophil. When phagocytosed, the particle turns from yellow/orange to red (neutral to alkaline pH), but when the particle is released by the neutrophil, it returns to its original color.
It is important to mention some of the limitations associated with using S-1. The fact that this dye has two emission spectra allowing ratiometric measurement is an advantage, but specialist equipment is needed to acquire images; the microscope used for the experiments must be able to record two images simultaneously or with an insignificant time delay. The authors assume that the researcher attempting this protocol has experience using confocal microscopy, or has access to a trained professional. We cannot list all the specific microscope parameters as they will differ for each microscope and need to be optimized by the researcher. The acetoxymethyl ester conjugated to S-1 that allows the dye to diffuse into the cell cytoplasm is degraded by non-specific esterases in the cell cytoplasm to form the fluorescent molecule. Esterases, such as alkaline phosphatase, are present in human serum and fetal bovine serum, which are used to supplement cell culture media. Accordingly, the medium in which the cells are loaded with S-1-AM (section 4.5) must not contain serum. This may prove challenging if using cells that require a more nutrient-rich medium to sustain them than the balanced salt solution used throughout this protocol. Similarly, other fluorescent medium components, such as phenol red, may interfere with S-1 measurements.
The error bars in Figure 3 indicate that there is some variation in the ratio measurements at each pH. A suitable number of repeats of each experiment (at least n = 3) and as many individual measurements in each single experiment are needed to overcome the inter-vacuolar variation. It is thus advisable to measure the pH of at least 100 phagosomes for each condition and as many phagosomal areas that appear to contain only one Candida. The phagosomes to be measured for quantitation should be those that have completely engulfed a Candida particle (i.e., those completely surrounded by cytoplasm). To mitigate against unintentional biases in the selection of cells/phagosomes for quantitation, all analyses should be performed while blind to the experimental conditions.
Here, we describe the isolation of neutrophils by dextran sedimentation of whole blood followed by centrifugation of the plasma layer through a density gradient. We use this technique as it quickly and efficiently produces a pure (&#62;95%) population of neutrophils, although there are other methods available, such as whole blood centrifugation through other density gradient formulas or negative selection of neutrophils using specialist kits with antibodies or magnetic beads. However, the latter can be prohibitively expensive for most groups who isolate neutrophils routinely. In addition, we use the anticoagulant heparin in the blood-collecting tube, whereas other researchers may be more accustomed to using ethylenediaminetetraacetic acid (EDTA) or acid citrate sodium (ACD). As there are many different methods to choose from, it is up to the personal preference of the researcher.
Furthermore, when isolating and manipulating neutrophils they should be handled with some care to avoid excessive activation. Precautionary steps include: only using plastic ware, no glass; filter-sterilize all buffers to remove any contaminating endotoxin; when spinning neutrophils make sure the centrifuge is well balanced to avoid excessive vibrations; limit as much as possible the time neutrophils remain in a pellet after centrifugation; do not maintain neutrophils in solution of more than 5 x 106/mL; and perform the experiment as soon as possible after isolation.
This method can be adapted to measure changes in the pH and phagosomal area over time by using a heated stage set to 37 &#176;C on the microscope and taking snapshots once every 30-60 s from the same position, as described for the calibration steps. It could also theoretically be adapted for higher-throughput experiments, such as in 96-well plates, and for flow cytometry experiments, where S-1 can be used as a pH indicator23. However, in these settings, the emphasis on individual cell activity is replaced by a more global effect on the cell population.
This method aims to provide a relatively simple experimental setup upon which individual researchers can adapt to suit their area of interest. Researchers may want to explore neutrophil phagosomal pH and area whilst also measuring movement of other ions, for example, intracellular calcium concentration. There are several fluorescent Ca2+ indicators readily available for confocal microscopy, such as Indo-1, which also has dual emission spectra at 400 and 475 nm24. These emission wavelengths do not overlap with S-1 emission spectra, but the excitation wavelength is at the ultraviolet (UV) end of the spectrum, which can be damaging to cells, and a UV laser is not commonplace on all microscopes. A comprehensive review of the different indicators to measure intracellular calcium flux is covered by Takahashi et al.25 and Hillson et al.26.
In conclusion, there are other methods available to measure phagosomal pH using different fluorescent dyes as Nunes et al. have demonstrated13 as well as other groups27,28. Other researchers have also used S-1 to measure cytosolic pH29 or phagosomal pH14. However, this protocol is unique in measuring simultaneously the pH of both cytosol and phagosome, which provides the opportunity to observe changes in phagosomal cross-sectional area and to distinguish between wildtype and Hvcn1-/- mouse neutrophils, and human neutrophils with and without a working NADPH oxidase.

נויטרופילים הם תא החיסון המולד בשכיחותו בגוף. תפקידה העיקרי שלו מורכב המסיירים הדם ואת ומלטפות, מעכלים את החלקיקים הזרים כי היא עשויה להיתקל בתהליך המכונה phagocytosis 1, 2. החלקיקים מפורקים בתא של תאיים שנקרא phagosome. הפעלת האנזים מונואמין NADPH נויטרופילים, את NOX2 איזופורם, יוזם מפל של תגובות ביוכימיות המסתיימת במותו של הפתוגן. יחידות משנה חלבון NOX2 להמשיך לגבש מורכבת שרשרת העברת אלקטרונים בקרום phagosomal 3. לאחר הפעלה, זה מעביר אלקטרונים מן NADPH דרך הממברנה לחמצן מולקולרי בתוך phagosome, הפקת אניוני דיסמוטאז ומיני חמצן תגובתי נוספת. תופעה זו ידועה בשם פרץ הנשימה, והוא נחשב חיוני הרג חיידקים יעיל בעיכול 2 </sup&gt;. עם זאת, תנועה בלעדית זו של מטען שלילי על פני הקרום שבקרוב להשבית NOX2 אם זה לא יפוצה על ידי מטען חיובי נעים ו / או מטען שלילי נע מתוך phagosome. זה הוכח גם כי הרוב המכריע של פיצוי תשלום נויטרופילים מתבצע על ידי ערוץ הפרוטון 4 HVCN1, 5. ערוץ זה מאפשר תנועה פסיבית של פרוטונים לאורך שיפוע אלקטרוכימיים שלהם מן cytosol לתוך phagosome. ריכוז Proton משתקף על ידי pH, כך על רמה מסוימת של פעילות אנזים מונואמין, מדידת pH של phagosome יכול לספק מידע על ההשתתפות היחסית של מסלולי protonic ולא protonic כפיצוי תשלום. Phagosome נויטרופילים האנושי יש pH בסיסי של כ 8.5 עבור 20-30 דקות לאחר phagocytosis 5. זה מרמז על קיומו של תעלות יונים הלא-פרוטונים נוספים NOX2-induced פיצוי תשלום, כמו פיוז&#39;ן ושחרור תכולתו של גרגרי חומצי ופיצוי בלעדי ידי HVCN1 ישמור בסביבת חומצית, בניגוד לזה שנצפה. התנועה של יונים לפצות מטען שלילי זה עשוי גם להפעיל שינויים בגודל phagosome באמצעות אוסמוזה. יונים יכול להיות אלה הנמצאים נויטרופילים בריכוזים פיסיולוגיים גבוהים: יוני אשלגן הוכחו לנוע לתוך phagosome 6, ותנועה יונית כלוריד היא עוד מועמד חשוב נויטרופילים פונקציה 7. הסדרת pH של phagosome חיונית עבור פעילות הפרוטאז מיקרוביאלית 5. Myeloperoxidase (MPO) נראה שיש פעילות אופטימלית ב- pH 6, בעוד שעבור cathepsin G ו אלסטט, רמות אופטימליות הן pH 7-9 ו pH 8-10, בהתאמה 5. לכן, שינוי חולף ב pH phagosomal עשוי לספק נישות פעילות אנזימים שונים כיףction. הבנה כיצד pH מעורב בהרג חיידקים נויטרופילים עשויה לספק מידע שימושי עבור העיצוב של סוכני מיקרוביאלית רומן משלים-נויטרופילים. Phagosome נויטרופילים היא סביבה מאוד תגובתי. זה עושה את זה קשה להעריך במדויק pH, בגלל צבע ניתן חמצון בקלות, מה שמוביל חפצים טכניים. מבחינה היסטורית, isothiocyanate והעמסת (FITC) כבר צבע של בחירה למדוד תאיים pH 8, 9. עם זאת, יש כמה חסרונות לשימוש שלה במדידת pH נויטרופילים phagosomal. יש לה pKa של 6.4 10, מה שאומר שזה יכול במדויק רק לשמש כדי להעריך את רמות מה PH 5 עד 7.5 8, כמו מרווה ב- pH &lt;8 11. כמו pH נויטרופילים phagosomal יכול להיות הרבה יותר אלקליין 5, FITC לא יכול ללכוד מגוון רחב של שינויים pH פוטנציאל. Signifi נוספתהבעיה צביעות עם FITC בהקשר של נויטרופילים היא שזה נחשב להיות photobleached ידי MPO 12. מעכב MPO, יזיד הנתרן, יכול לשמש כדי להגביל photobleaching 13, אבל זה הוכח כי יזיד הנתרן ישירות מוריד את רמת חומציות phagosomal באופן MPO עצמאי ולכן היא בלתי הולמת לשימוש מבחנים כגון 5. לעומת צבעי תאיים אחרים, S-1 יש pKa גבוה יחסית של 7.5 10. בתנאים חומציים, המולקולה היא protonated ומייצרת אות פליטה בין 560 ו 600 ננומטר כאשר נרגש על 488 ננומטר ומעלה. כאשר המולקולה היא deprotonated בלמעלה תנאי בסיסי, גל הפליטה נגמר 600 ננומטר. יחס של עוצמות הקרינה בשני אורכי גל אלה מצביע על שינוי הפליטה, המהווה יותר הוא אמין יותר מאשר מדידות קרינה יחידות, כפי שהוא אינו מושפע על ידי ריכוז fluorophore ואת מבנה תא. S-1 ניתן מצומדות חומר אנטיגני, כגון zymosan 14, אם כי-הרג חום (HK) קנדידה אלביקנס היא מועדף, שכן שטח הפנים הגדול יותר נותן קריאת קרינה עקבית יותר. השתמשנו גם שינוי של השיטה בחקר שינויים זמניים ב pH (איור 3) 5. שיטה זו למדידת pH cytosolic ניתן ליישם בקלות סוגי תאים אחרים, כמתואר במקום אחר 15, 16, ו תאים עם phagosomes אלקליין יותר 14.

<table border="0" cellpadding="0" cellspacing="0" width="851">
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		<col />
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Candida albicans ATCC 10231 Vitroids&#160; 80 CFU</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">RQC14003-10EA</td>
			<td style="width:360px;">Place directly on a spread agar plate according to the manufacturer&#39;s instructions</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">YPD broth</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">Y1375</td>
			<td style="width:360px;">Follow manufacturer&#39;s instructions, various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Dextran Clinical grade MW = 200,000-300,000</td>
			<td style="width:99px;">MP Biomedicals</td>
			<td style="width:119px;">2101514</td>
			<td style="width:360px;">Use gloves&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Heparin Sodium solution for infusion (preservative free), 1,000IU/ml&#160;</td>
			<td style="width:99px;">Fannin, Wockhardt UK Ltd</td>
			<td style="width:119px;">FP1077</td>
			<td style="width:360px;">We purchased from UCH inpatient pharmacy</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">SNARF-1 Carboxylic Acid, Acetate, Succinimidyl Ester - Special Packaging</td>
			<td style="width:99px;">Thermo Scientific/Invitrogen</td>
			<td style="width:119px;">SS22801</td>
			<td style="width:360px;">Use gloves</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:99px;">Thermo Scientific/Invitrogen</td>
			<td style="width:119px;">C1272</td>
			<td style="width:360px;">Use gloves</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Vivaglobin human IgG&#160; 160mg/ml solution</td>
			<td style="width:99px;">CSL Behring</td>
			<td style="width:119px;">received as a gift</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Normal mouse serum</td>
			<td style="width:99px;">Abcam</td>
			<td style="width:119px;">ab7486</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Lymphoprep (density gradient medium)</td>
			<td style="width:99px;">Alere Ltd</td>
			<td style="width:119px;">1114547</td>
			<td style="width:360px;">Keep sterile and away from direct sunlight, can be substituted by Ficoll-Paque</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Nigericin</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">N7143</td>
			<td style="width:360px;">Toxic, use gloves, various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Saponin</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">47036</td>
			<td style="width:360px;">Toxic, use gloves, various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Dimethyl sulphoxide (DMSO)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td style="width:360px;">Toxic, use gloves, various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503&#160;</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Potassium chloride (KCl)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Sodium chloride (NaCl)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Magnesium chloride (MgCl2)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Magnesium sulphate (MgSO4)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Potassium dihydrogen phosphate (KH2PO4)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9791</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Calcium chloride (CaCl2)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Glucose</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9434</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Absolute Ethanol&#160;</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">2860</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Diphenylene iodonium (DPI)</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">D2926</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Zinc chloride</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">208086</td>
			<td style="width:360px;">Various providers exist, do not dilute in phosphate based buffers as it will precipitate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Poly L lysine</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">P4707</td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Laser scanning confocal microscope (Zeiss LSM 700)</td>
			<td style="width:99px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:360px;">The microscope must be able to excite at 555nm and measure emission at two channels simultaneously (560-600nm, &#62;610nm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Ibidi &#181; slide 8 well ibiTreat</td>
			<td style="width:99px;">Thistle Scientific</td>
			<td style="width:119px;">IB-80826</td>
			<td style="width:360px;">Other appropriate imaging dishes can be substituted</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Fluorodish Cell Culture Dish-35mm, 23mm well</td>
			<td style="width:99px;">World Precision Instruments&#160;</td>
			<td style="width:119px;">FD35-100</td>
			<td style="width:360px;">Other appropriate imaging dishes can be substituted, different sizes are available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Soniprep 150</td>
			<td style="width:99px;">MSE</td>
			<td style="width:119px;"></td>
			<td style="width:360px;">Any appropriate sonicator will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">TC20 Automated Cell Counter</td>
			<td style="width:99px;">BioRad</td>
			<td style="width:119px;"></td>
			<td style="width:360px;">Various providers exist</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">1.5ml Protein LoBind Tubes</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z666505-100EA</td>
			<td style="width:360px;">Specially used as Candida can stick to normal eppendorfs</td>
		</tr>
	</tbody>
</table>

imaging the neutrophil phagosome and cytoplasm using a ratiometric ph indicator

נויטרופילים הם קריטיים לארח הגנת מולד, וכתוצאה מכך, מהווים תחום חשוב של מחקר רפואי. Phagosome, התא התאי שבו ההרג והעיכול של חלקיקים הציפו להתקיים, הוא הזירה העיקרית הרג הפתוגן נויטרופילים הדורש רגולציה הדוקה. Phagosomal pH הוא היבט אחד כי נשלט בקפידה, בתורו מסדיר פעילות הפרוטאז מיקרוביאלית. רבים ניאון צבעי pH רגיש שימשו כדי להמחיש את הסביבה phagosomal. יש S-1 מספר יתרונות על פני צבעי pH אחרים רגיש, כוללים ספקטרום הפליטה הכפול שלה, עמידותו-הלבנת תמונה, ואת pKa הגבוהה שלה. באמצעות שיטה זו, אנו הוכחנו כי phagosome נויטרופילים הוא אלקליין חריג בהשוואת פגוציטים אחרים. באמצעות הטיות ביוכימיים שונים של הצבע, phagosome ניתן שמסומן מן הציטופלסמה כך ששינויים בגודל ובצורה של phagosome ניתן להעריך. זה מאפשר Fאו ניטור נוסף של תנועה יונית.

Figure 1 presents snapshots of neutrophils from different origins to demonstrate varying phagosomal environments. To ease quantitative analysis, it is important to seed the wells with an appropriate number of cells: too many will cause the cells to layer over each other, making it difficult to view enclosed phagosomes accurately; too few will, of course, provide fewer results, particularly as not every neutrophil will phagocytose. Figure 2 is an image that is over-saturated; this can be assessed by splitting the image between its two channels (using the microscope software recommended in the Materials List or an equivalent) &#8212; red dots show where maximum fluorescence has been detected. This can be countered by reducing the intensity of the laser. Calibration curves using the various buffer systems are shown in Figure 3, adapted from Levine et al.5. The error bars show that there is some variation in fluorescence between readings. Figure 4 gives an example of how the data for phagosomal pH and area could be presented. This approach allows each individual measurement to be displayed with an over-laying boxplot. However, the data could also be displayed in a histogram bar chart.
<img alt="Figure 1" src="/files/ftp_upload/55107/55107fig1.jpg" /> 
Figure 1: Appropriate snapshot images of neutrophils from humans and mice. To the far right is a qualitative visual key of the approximate color of the S-1-stained phagosomes corresponding to the pH. The yellow color indicates more acidity, while red indicates more alkalinity. (A) shows Hvcn1-/- mouse bone marrow neutrophils 20 min after phagocytosis. The phagosomes appear very red, alkaline, and swollen. The red arrow in the bottom right part of the image points to an intracellular Candida, while the arrow in the top left points to an extracellular particle. (B) shows wildtype mouse bone marrow neutrophils that have ingested Candida; they are much less alkaline than Hvcn1-/- cells. (C) shows human peripheral blood neutrophils at the same point after phagocytosis. They appear slightly more alkaline than the mouse wildtype cells, but the phagosomes are still not as large and red as the Hcvn1-/- cells. (D) shows human neutrophils that have phagocytosed Candida in the presence of 5 &#181;M diphenylene iodonium (DPI). All the phagosomes are very acidic, with a pH of 6 or less; the drug inhibits the NADPH oxidase, so there is no compensatory ion movement. The protons released from the acidic granules that fuse to the phagosome cause the acidic pH20, and increased recruitment of the V-ATPase to the phagosomal membrane upon treatment with DPI9. The cytoplasm also appears more alkaline compared to the cells from the other conditions. <a href="http://cloudflare.jove.com/files/ftp_upload/55107/55107fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55107/55107fig2.jpg" /> 
Figure 2: Over-saturation of Hvcn1-/- mouse bone marrow neutrophils. It is important to exclude from the analysis images in which the fluorescence data are over-saturated. As described in section 5.3, the composite image is split into two images (top left, red arrow) with both channels presented individually. In this software, range indicator is checked on (bottom left, red arrow), then any pixels which are over-saturated are bright red. There is over-saturation present in both channels (1 and 2). A magnification of some cells and extracellular Candida is shown at the bottom right, with arrows pointing to affected areas. Analyzing these points would lead to a false ratiometric measurement. <a href="http://cloudflare.jove.com/files/ftp_upload/55107/55107fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55107/55107fig3.jpg" /> 
Figure 3: Standard calibration curves for the conversion of S-1 fluorescence ratios to pH measurements. The standard curves for Candida alone in the different buffers (labeled as &#34;Tris&#34;) and when the cells are permeabilized with saponin (&#34;Saponin&#34;) are very similar, showing that the S-1 reading inside and outside the cell (phagosome and extracellular medium) are comparable. The error bars represent the mean &#177; SD. The S-1 ratio/pH curve is shortened when tested in the cytoplasm (&#34;Cytoplasm&#34;), which should be taken into consideration in the image analysis. This figure has been modified from Levine et al.5. <a href="http://cloudflare.jove.com/files/ftp_upload/55107/55107fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55107/55107fig4.jpg" /> 
Figure 4: Quantification of phagosomal pH and area. This figure presents an example of data presentation. The graphs were generated using the programming software R. A: shows phagosomal ratio and corresponding pH for A (Hvcn1-/- mouse bone marrow neutrophils), B: wildtype mouse bone marrow neutrophils, C: human peripheral blood neutrophils, and D: human neutrophils with 5 &#181;M DPI n = 3/300. Individual measurements are shown as small squares, with an overlaying boxplot with median and interquartile range. A red bar represents the mean. As seen in the images in Figure 1, Hvcn1-/- cells have very alkaline phagosomes in comparison to wildtype mouse and human neutrophils. They also have a bigger phagosomal area (Figure 4B, n = 3/300). Human neutrophil phagosomes are slightly more alkaline and larger than wildtype mouse neutrophils, while human neutrophils incubated with DPI have very acidic and small phagosomes. <a href="http://cloudflare.jove.com/files/ftp_upload/55107/55107fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table>
	<tbody>
		<tr>
			<td colspan="4">Balanced salt solution (BSS) buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>156 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>3 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>2 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>1.25 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>2 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>10 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>10 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">pH 7.4 with NaOH or HCl</td>
		</tr>
		<tr>
			<td colspan="4">YPD broth</td>
		</tr>
		<tr>
			<td>YPD broth</td>
			<td>50 g</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>1 L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Autoclave at 121 &#176;C for 15 min</td>
		</tr>
		<tr>
			<td colspan="4">For YPD agar: before autoclaving add 15 g/L agar</td>
		</tr>
		<tr>
			<td colspan="4">10% Dextran solution</td>
		</tr>
		<tr>
			<td>Dextran clinical grade</td>
			<td></td>
			<td>50 g</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td>4.5 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td>500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Add to glass bottle and autoclave at 121 &#176;C for 15 min</td>
		</tr>
		<tr>
			<td colspan="4">2x Saline solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td>18 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water&#160;</td>
			<td></td>
			<td>1 L</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Add to glass bottle and autoclave at 121 &#176;C for 15 min</td>
		</tr>
		<tr>
			<td colspan="4">10% Saponin stock</td>
		</tr>
		<tr>
			<td>BSS buffer</td>
			<td></td>
			<td>50 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td></td>
			<td>5 g</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Heat BSS buffer to 37 &#176;C, add saponin and mix.</td>
		</tr>
		<tr>
			<td colspan="4">Add 0.1% sodium azide as preservative, store mixture at 4 &#176;C.</td>
		</tr>
		<tr>
			<td colspan="4">Calibration buffers</td>
		</tr>
		<tr>
			<td colspan="4">Candida standard curve</td>
		</tr>
		<tr>
			<td colspan="4">First make up 0.15 M stock of each buffer</td>
		</tr>
		<tr>
			<td colspan="4">Make up 0.15 M NaCl solution</td>
		</tr>
		<tr>
			<td colspan="4">15 mL final volume: 5 mL 0.15 M of desired buffer solution + 10 mL 0.15 M NaCl solution</td>
		</tr>
		<tr>
			<td>pH 3</td>
			<td>100 mM NaCl</td>
			<td>50 mM glycine</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 4</td>
			<td>100 mM NaCl</td>
			<td>50 mM acetate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 5</td>
			<td>100 mM NaCl</td>
			<td>50 mM acetate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 6</td>
			<td>100 mM NaCl</td>
			<td>50 mM acetate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 7</td>
			<td>100 mM NaCl</td>
			<td>50 mM Tris</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 8</td>
			<td>100 mM NaCl</td>
			<td>50 mM Tris</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 9</td>
			<td>100 mM NaCl</td>
			<td>50 mM Tris</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 10</td>
			<td>100 mM NaCl</td>
			<td>50 mM glycine</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 11</td>
			<td>100 mM NaCl</td>
			<td>50 mM phosphate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 12</td>
			<td>100 mM NaCl</td>
			<td>50 mM phosphate</td>
			<td></td>
		</tr>
		<tr>
			<td>pH 13</td>
			<td>100 mM NaCl</td>
			<td>50 mM phosphate</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cytosolic standard curve</td>
		</tr>
		<tr>
			<td colspan="4">Use previously made 0.15 M stock buffer solutions</td>
		</tr>
		<tr>
			<td colspan="4">Make up 0.15 M KCl solution</td>
		</tr>
		<tr>
			<td colspan="4">15 mL final volume: 5 mL 0.15 M of desired buffer + 10 mL 0.15 M KCl solution</td>
		</tr>
		<tr>
			<td colspan="4">10 mM stock of nigericin in ethanol, add 15 &#181;L to each final volume solution</td>
		</tr>
		<tr>
			<td>pH 3</td>
			<td>100 mM KCl</td>
			<td>50 mM glycine</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 4</td>
			<td>100 mM KCl</td>
			<td>50 mM acetate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 5</td>
			<td>100 mM KCl</td>
			<td>50 mM acetate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 6</td>
			<td>100 mM KCl</td>
			<td>50 mM acetate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 7</td>
			<td>100 mM KCl</td>
			<td>50 mM Tris</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 8</td>
			<td>100 mM KCl</td>
			<td>50 mM Tris</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 9</td>
			<td>100 mM KCl</td>
			<td>50 mM Tris</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 10</td>
			<td>100 mM KCl</td>
			<td>50 mM glycine</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 11</td>
			<td>100 mM KCl</td>
			<td>50 mM phosphate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 12</td>
			<td>100 mM KCl</td>
			<td>50 mM phosphate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td>pH 13</td>
			<td>100 mM KCl</td>
			<td>50 mM phosphate</td>
			<td>10 &#181;M nigericin</td>
		</tr>
		<tr>
			<td colspan="4">pH all calibration solutions with either HCl or NaOH</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of buffers. This table describes the appropriate compositions of the different buffers used in the protocol.

Supplementary code file. This file contains a number of macros, written by A. P. Levine, which are necessary for image analysis. The authors would be happy to try to address any queries associated with using this code. <a href="https://www.jove.com/files/ftp_upload/55107/Phagosomal_measurement.txt" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator at JoVE.com

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

הדמיה נויטרופילים Phagosome ו הציטופלסמה באמצעות אינדיקטור pH ratiometric

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

imaging-neutrophil-phagosome-cytoplasm-using-ratiometric-ph

Research

JoVE Journal

Immunology and Infection

10.0K Views.  University College London. This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Video: Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

באמצעות הדמיה Bioluminescent לחקור סינרגיזם בין Streptococcus pneumoniae ו שפעת וירוס עכברים תינוקות

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

דימות אופטי לא פולשנית של כלי דם הלימפה של עכבר

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

נדידת תאים דנדריטים ניטור באמצעות<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H דימות תהודה מגנטית

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

חקר Phagolysosome Biogenesis בהמקרופאגים בשידור חי

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

מדידת pH Phagosome ידי ratiometric מיקרוסקופ פלואורסצנטי

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

הדמית ratiometric של pH התאי Biofilms השיניים

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

הדמיה Intravital של Priming נויטרופילים שימוש IL-1β מונחה יזם עכברים Reporter DsRed

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Assay סגירת Phagosome&quot; לדמיין גיבוש Phagosome בשלושה ממדים באמצעות סך פנימית השתקפות מיקרוסקופ פלואורסצנטי (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

פיתוח זיהוי של subpopulation רומן של אדם נויטרופילים הנגזר הענק Phagocytes<em&gt; במבחנה</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

הדמיה נויטרופילים Phagosome ו הציטופלסמה באמצעות אינדיקטור pH ratiometric

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באמצעות הדמיה Bioluminescent לחקור סינרגיזם בין Streptococcus pneumoniae ו שפעת וירוס עכברים תינוקות

דימות אופטי לא פולשנית של כלי דם הלימפה של עכבר

נדידת תאים דנדריטים ניטור באמצעות

חקר Phagolysosome Biogenesis בהמקרופאגים בשידור חי

מדידת pH Phagosome ידי ratiometric מיקרוסקופ פלואורסצנטי

הדמית ratiometric של pH התאי Biofilms השיניים

הדמיה Intravital של Priming נויטרופילים שימוש IL-1β מונחה יזם עכברים Reporter DsRed

"Assay סגירת Phagosome" לדמיין גיבוש Phagosome בשלושה ממדים באמצעות סך פנימית השתקפות מיקרוסקופ פלואורסצנטי (TIRFM)

פיתוח זיהוי של subpopulation רומן של אדם נויטרופילים הנגזר הענק Phagocytes

מעקב אחר תמציות ביומיסקופיה באמצעות מיקרוסקופ Confocal וקדי