The authors are financially supported by the Swiss National Science Foundation through an operating grant N&#176; 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).

הצהרת אתיקה: כל המניפולציות של בעלי החיים בוצעו בהתאם להנחיות קפדניות של ועדת המחקר בבעלי חיים של אוניברסיטת ג&#39;נבה. 1. הכנת יעדי phagocytic <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 20 מ&quot;ג של zymosan המיובש 10 מיליליטר שנאגרו מלוח פוספט סטרילי (PBS). מערבולת וחום באמבט מים רותח למשך 10 דקות. מגניב ו צנטריפוגות XG ב 2000 במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> הסר את supernatant, resuspend ב 1 מיליליטר PBS וsonicate במשך 10 דקות באמבט המים sonicator. העבר את 500 μl לשני צינורות 1.5 מיליליטר. צנטריפוגה ב 11,000 XG במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> חזור על לשטוף עם 500 μl PBS. Zymosan מבושל יכול להיות aliquoted, קפוא ומאוחסן ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> לשטוף zymosan פעמיים ב500 μl מוכן טרי 0.1 M Na 2 CO 3, pH 9.3 כאמור לעיל (שלב 1.2). </li><li style=";text-align:right;direction:rtl"> גלול ב490 μl לאחר השטיפה הסופית. הוסף 10 μl של 10 מ&quot;ג / מ&quot;ל ​​מומס FITC בsulfoxid דימתילדואר (DMSO), מערבולת, מכסה בנייר כסף, ודגירה עם תסיסה במשך 4 שעות ב RT. </li><li style=";text-align:right;direction:rtl"> כדי להסיר צבע מאוגד, לשטוף כאמור לעיל (שלב 1.2), ראשון עם 0.5 מיליליטר DMSO + 150 μl PBS, אז 0.5 מיליליטר DMSO + 300 μl PBS, אז 0.5 מיליליטר DMSO + 450 μl PBS, אז PBS לבד. </li><li style=";text-align:right;direction:rtl"> רוזן zymosan באמצעות haemocytometer. הוסף 1 μl של zymosan 500 μl PBS, מערבולת, ולאחר מכן להוסיף 10 μl לקצה תא haemocytometer, שבו הוא פוגש את coverslip הניח על הרשת המרכזית. הר haemocytometer על מיקרוסקופ שדה בהיר מצויד מטרת 20X. </li><li style=";text-align:right;direction:rtl"> להתמקד בפינה השמאלית העליונה מכילה 16 ריבועים ולספור את כל חלקיקי zymosan בתחום זה באמצעות דלפק יד נקודות. חזור עם הפינה הימנית התחתונה. חשב את ריכוז zymosan באמצעות המשוואה הבאה: ריכוז Zymosan = רוזן / 2 x 500 x 10 4 zymosan / מיליליטר. zymosan שכותרתו ניתן aliquoted, קפוא ומאוחסן ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> Opsonize Wi zymosan הכותרתth נוגדן אנטי zymosan ארנב ב 1: 100 דילול. מערבולת דגירה עבור שעה 1 באמבט מים 37 מעלות צלזיוס. לשטוף עם 500 μl PBS כאמור לעיל (שלב 1.2). הערה: Sonication מומלץ מאוד, במיוחד לאחר opsonization, כzymosan נוטה ליצור גושים שיכולים לעשות ניתוח קשה יותר בהמשך. Opsonization עם 65% חולדה V / V או סרום אדם עשוי לשמש כחלופה. </li><li style=";text-align:right;direction:rtl"> רוזן zymosan באמצעות haemocytometer כפי שמתואר בשלבים 1.7-1.8, ולדלל עד 100 x 10 6 מיליליטר / חלקיקים. Opsonization עם גירוי phagocytic אחר, כגון השלמה או לא opsonization, ניתן לבצע לחלופין. </li></ol> 2. חיים מיקרוסקופית וידאו <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבודד נויטרופילים עכבר עיקריים כפי שתואר בפירוט במקום אחר 38. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחלופין, להשתמש בכל סוג תא phagocytic. שמרו נויטרופילים על קרח ברוזוול פרק זיכרון המכון (RPMI) מדיום 1640 בתוספת 5% בסרום עגל עוברי (V / V), 200 פניצילין U / streptomycin, בריכוז של 10 x 10 6 תאים / מיליליטר עד מוכן לשימוש. סוגי תאים אחרים יכולים להיות שנזרעו 1-4 ימים לפני. </li></ol></li><li style=";text-align:right;direction:rtl"> הוסף 2 x 10 5 (20 μl) של נויטרופילים למרכז צלחת זכוכית תחתונה 35 מ&quot;מ, להפיץ את הירידה על ידי הוספת 50 μl של מדיום ולדגור על 37 מעלות צלזיוס למשך 5 דקות כדי לאפשר לתאים לדבוק. </li><li style=";text-align:right;direction:rtl"> הוסף 1 מיליליטר של תמיסת מלח המאוזן של האנק (HBSS) חימם עד 37 ° C המכיל hydrazide מעכב 4-aminobenzoic MPO הספציפי (4-ABH, 10 מיקרומטר). גם מעכבי MPO שאינם ספציפיים, כגון אזיד הנתרן (5 מ&quot;מ), ניתן להשתמש אך הוצעו לאחרונה להפעיל אפקטים שאינם ספציפיים נוספים על pH 39. </li><li style=";text-align:right;direction:rtl"> הר הצלחת על מיקרוסקופ פלואורסצנטי widefield מצויד במערכת חימום 37 ° C ואובייקטיביים שמן 40X. דגירה תאים בלי הדמיה במשך 10 דקות כדי לאפשר לתאים וציוד לאזן. </li><li style=";text-align:right;direction:rtl"> התאם את הגדרות מיקרוסקופ כדי לרכוש תמונת שידור בהיר שדה [PHAse או התערבות ההפרש לעומת זאת (DIC) אם קיימים] עם 440 ננומטר או 490 ננומטר עירור ופליטת 535 ננומטר כל 30 שניות. </li><li style=";text-align:right;direction:rtl"> התאם זמן חשיפה ותדירות רכישה בהתאם למערכת מיקרוסקופ כדי למנוע photobleaching יותר מדי וphototoxicity ובהתאם לשאלות הניסיוניות שהתבקש. בגין רכישת תמונה. </li><li style=";text-align:right;direction:rtl"> אחרי 2 דקות, להוסיף 10 x 10 6 (50 μl) opsonized FITC-zymosan למרכז הצלחת. התאם יעד: יחסי תא בהתאם לסוג של תָא בַּלעָן ויעד בשימוש. </li><li style=";text-align:right;direction:rtl"> המשך הדמיה למשך 30 דקות. לאחר 30 דקות להפסיק את רכישת הזמן לשגות ולהתחיל טיימר. הזז את הבמה וללכוד 10 או יותר תמונות בתחומים שונים של נוף לphagosomes אחר התמונה, כל זאת במסגרת 5 דקות. </li></ol> 3. ניסויי כיול והבקרה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את פתרונות כיול pH (מתכונים מתוארים בטבלה 1) לפני היום של ניסוי ולהקפיא בaliquots 10 מיליליטר.להפשיר את פתרונות כיול וחמים עד 37 מעלות צלזיוס באמבט מים. בדוק את ה- pH עם מד pH ולהקליט את ה- pH בפועל של פתרונות. </li><li style=";text-align:right;direction:rtl"> הר משאבת peristaltic על צלחת תחתית הזכוכית לפני רכישת תמונה ולבצע מיקרוסקופיה וידאו החי כפי שתואר לעיל (סעיף 2). </li><li style=";text-align:right;direction:rtl"> בתום תקופת הרכישה 30 דקות להפוך את המשאבה בלהסיר את הפתרון בתוך הצלחת, להוסיף 1 מיליליטר של פתרון הכיול הראשון ולעצור את המשאבה. </li><li style=";text-align:right;direction:rtl"> חכה עד שהאות תתייצב, בדרך כלל 5 דקות. חזור עם כל פתרון כיול. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבצע כיול לפחות אחת ליום ניסיוני, ולכייל מתחיל עם pH הגבוה ביותר ועובד ברצף לרמה הנמוכה ביותר, כמו גם החל מה- pH הנמוך ביותר ועובד ברצף לקראת הגבוה ביותר. </li></ol></li><li style=";text-align:right;direction:rtl"> כביקורת, להוסיף של יודיד propionium diphenyl מעכבי מונואמין NADPH 100 מיקרומטר 100 μl (DPI) בדילול מלא בHBSS לתאים ב900 μl HBSS בצלחת תחתית הזכוכית, ודגירה של 10 דקות. </li><li style=";text-align:right;direction:rtl"> התחל רכישה ולהוסיף zymosan כאמור לעיל (שלב 2.7). לאחר 15 דקות של phagocytosis, להוסיף 10 μl של 10 מיקרומטר מעכב V-ATPase concanamycin (קונקה) בדילול מלא HBSS ולהמשיך הדמיה ל-15 דקות נוספות. </li></ol> 4. ניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ניתוח של הזמן לשגות סרטים ותמונות phagosome. הערה: ההוראות הבאות הן ספציפיות לImageJ. צעד אחר צעד הוראות עשויות להשתנות במידה רבה בהתאם לתוכנה בשימוש, אבל הפונקציות מתוארות בסעיף זה זמינות ברוב תוכנות ניתוח תמונה מקצועיות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פתח את התמונות עם תוכנת ניתוח תמונה מקצועית. הפחת את הרקע בערוץ 490 על ידי ציור ROI כיכר קטן עם כלי בחירת מצולע מרובעים באזור שבו אין תאים, ולחיצה חיסור התוספים&gt; BG מהחזר על ההשקעה. לאחר מכן להעלות אותו ההחזר על ההשקעה בערוץ 440 בעריכה&gt; בחירת לחיצה&gt; שחזור בחירה וחזור. </li><li style=";text-align:right;direction:rtl">הפוך תמונת יחס 32 סיביות של הערוץ 490 מחולק בערוץ 440 על ידי לחיצה על תהליך&gt; מחשבון תמונה ..., בחירת התמונות המתאימות בתפריטים הנפתחים Image1 וImage2, ולאחר מכן בחירה &quot;הפרד&quot; בתפריט הנפתח מבצע , ובדיקה של 32 סיביות (לצוף) תיבת סימון תוצאה. </li><li style=";text-align:right;direction:rtl"> שנה את הטבלה להסתכל למעלה קידוד הצבע לצבע-קידוד יחס תואם על ידי לחיצה שולחנות תמונה&gt; בדיקה&gt; קשת RGB. סף את תמונת היחס לחסל 0 ואינסוף פיקסלים על ידי לחיצה תמונה&gt; התאם&gt; סף .... התאם את פסי הגלילה כך zymosan שמופיע באדום ולחץ על החל, ולוודא פיקסלים רקע הסט לתיבת סימון נאן הוא נבחר. </li><li style=";text-align:right;direction:rtl"> לשלב ערימת יחס זה עם הערוץ הבהיר שדה למרוכבים רב-ערוצים על ידי לחיצה תמונה&gt; צבע&gt; מיזוג ערוצים, ובחירת התמונה הבהיר השדה בתפריט הנפתח ערוץ האפור ותמונת היחס בתפריט הנפתח הערוץ הירוק, ו בחירהשמור את תמונות מקור תיבת סימון. סרוק את הזמן לשגות סרטים או תמונות כדי לקבוע אילו phagosomes הן להיות מנותח. הערוץ בהיר השדה הוא שימושי עבור זה. </li><li style=";text-align:right;direction:rtl"> לצייר אזורים של העניין (ROI) באמצעות כלי בחירת מצולע הסגלגלים סביב phagosomes, ולהוסיף אותם למנהל את ההחזר על ההשקעה על ידי לחיצה לנתח&gt; כלים&gt; מנהל ROI&gt; להוסיף. למדוד יחס עוצמת zymosan על ידי לחיצה נוסף&gt; Multi-מדוד ודה-בחירת השורה אחת לכל תיבת סימון פרוסה. </li><li style=";text-align:right;direction:rtl"> לזמן לשגות סרטים, מסלול phagosomes אחד בכל פעם על ידי ציור את ההחזר על ההשקעה, הקלדת Ctrl + M כדי למדוד כל נקודת זמן, ונע ההחזר על ההשקעה בעת צורך לעקוב אחר ערכי עוצמה לאורך זמן. העתק את המדידות מחלון התוצאות לתוכנת גיליון אלקטרוני. הערה: ניתוח של 15 (5 קורסי זמן לניסוי x 3) ניסויים בלתי תלויים היא אידיאלית, אבל פחות או יותר עשוי להידרש בהתאם להשתנות נצפתה. שמירת ROIs תמיד מומלץ. כמה חבילות תוכנה מאפשרות Regi תמונהstration ועדכון דינמי של ROIs, להקל בחלק זה של הניתוח. </li></ol></li><li style=";text-align:right;direction:rtl"> המרה מקרינה לערכי pH <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מדוד את היחס 490/440 לphagosomes בזמן לשגות סרטי הכיול, כמתואר בסעיף 4.1. </li><li style=";text-align:right;direction:rtl"> בגיליון אלקטרוני, להתוות ערכי היחס לאורך זמן, ולחשב את ערכי יחס הממוצע מכל phagosomes בתוך שדה הראייה (בדרך כלל, בין 5-20) מ -5 נקודות זמן בתוך אמצע תקופת הזמן המתאים לכל כיול pH פִּתָרוֹן. (ראה גם את התיבות האדומות באיור 5 א.) </li><li style=";text-align:right;direction:rtl"> עלילה ערכים אלה ממוצע 490/440 יחס נגד pH נמדד בפועל. לשלב לפחות 3 כיולים עצמאיים לגרף אחד. השתמש בחבילת תוכנה סטטיסטית או מספרית לבצע רגרסיה ליניארית (עקומה בכושר הטובה ביותר), בדרך כלל למשוואת sigmoidal. הערה: לדוגמא, באיור 5 המשוואה השתמשה הייתה sigmoidal ולצמן [Y = + תחתון ((למעלה-למטה)/ (1 + EXP ((V50-X) / Slope))], שבו ערכי Y הם 490/440 יחסים, X הם ערכי pH ולמעלה, למטה, פרמטרים V50 ומדרון מחושבים על ידי התוכנה. </li><li style=";text-align:right;direction:rtl"> השתמש במשוואה שהתקבלה להפוך ערכי יחס לpH. הערה: לדוגמא, באיור 5 המשוואה שהתקבלה עבור עקומת הכיול בנוכחות אזיד הנתרן היא 490/440 יחס = 1.103 + [(2.89-1.103) / (1 ​​+ EXP ((6.993-pH) /0.9291)] . פתרון לpH נותן את המשוואה הבאה: pH = 6.993-.9291 * [LN ((1.178 / (יחס-1.103)) - 1))]. </li></ol></li></ol>

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The authors have nothing to disclose.

למרות זמן רב יותר מאשר שיטות חלופיות, כגון ספקטרוסקופיה וFACS, אשר מעסיקות אסטרטגיה דומה של שימוש בצבע רגיש pH מצמידים את המטרות אבל למדוד את ה- pH הממוצע של אוכלוסיית phagosomes, מיקרוסקופיה מציעה מספר יתרונות. הראשון הוא שפנימי וחיצוני קשורים, אבל לא הפנים, חלקיקים יכולים בק?...

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2- and its derivative reactive oxygen species (ROS) 2,4. 
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl-/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33. 
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses. 
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

<table>
	<tbody>
		<tr>
			<td>Zymosan A powder</td>
			<td>Sigma-Aldrich</td>
			<td>Z4250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)</td>
			<td>Life Technologies</td>
			<td>Z2850</td>
			<td>Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.</td>
		</tr>
		<tr>
			<td>4-Aminobenzoic hydrazide (4-ABH)</td>
			<td>Santa Cruz</td>
			<td>sc-204107</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Diphenyleneiodonium chloride (DPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D2926</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Concanamycin A (ConcA)</td>
			<td>Sigma-Aldrich</td>
			<td>27689</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma-Aldrich</td>
			<td>N7143</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Monensin</td>
			<td>Enzo</td>
			<td>ALX-380-026-G001</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14200-075</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Hank&#39;s balance salt solution</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td>Ringer&#39;s balanced salt solution or other clear physiological buffers may be substituted.</td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>N-Methyl-D-glucamine (NMDG)</td>
			<td>Sigma-Aldrich</td>
			<td>M2004&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>3777</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>&#160;Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Sigma-Aldrich</td>
			<td>T1503&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (EtOH)</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Glass-bottom 35 mm petri dishes (Fluorodish)</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td>Ibidi &#181;-clear dishes or coverslips with appropriate imaging chambers may be sustituted</td>
		</tr>
		<tr>
			<td>Sonicating water bath</td>
			<td>O. Kleiner AG</td>
			<td></td>
			<td>A sonicator may be used instead, various instrument providers exist</td>
		</tr>
		<tr>
			<td>Heamocytometer</td>
			<td>Marienfeld GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Widefield live imaging microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Dynamax RP-1)</td>
			<td>Rainin</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Schott Gerate GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Manual Counter</td>
			<td>Milian SA</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
	</tbody>
</table>

measuring phagosome ph by ratiometric fluorescence microscopy

Phagocytosis הוא תהליך בסיסי שדרכו תאים חיסוניים מולדים לבלוע חיידקים, תאים אפופטוטיים או חלקיקים זרים אחרים כדי להרוג או לנטרל את חומר בליעה, או להציג אותה כאנטיגנים וליזום תגובה חיסונית אדפטיבית. PH של phagosomes הוא פרמטר קריטי המסדיר ביקוע או היתוך עם endomembranes והפעלה של אנזימי מפרקי חלבונים, אירועים המאפשרים vacuole phagocytic להתבגר לתוך אברון נגרע. בנוסף, טרנסלוקציה של H + נדרשת לייצור רמות גבוהות של מינים תגובתי חמצן (ROS), שהם חיוניים להרג יעיל ואיתות לרקמות מארח אחרות. פתוגנים תאיים רבים לחתור תחת הרג phagocytic ידי הגבלת החמצת phagosomal, המדגיש את חשיבותו של ה- pH בביולוגיה phagosome. כאן אנו מתארים שיטה למדידת pH ratiometric phagosomal בנויטרופילים באמצעות isothiocyanate והעמסת (FITC) -labeled zymosan כטארג phagocyticETS, וLive- תא הדמיה. Assay מבוסס על תכונות הקרינה של FITC, שהוא הרווה ידי pH חומצי כאשר נרגש ב 490 ננומטר, אך לא כאשר נרגש ב 440 ננומטר, המאפשר כימות של יחס pH תלוי, ולא הקרינה מוחלטת, של צבע אחד. פרוטוקול מפורט לביצוע בכיול צבע אתר והמרה של יחס לערכי pH האמיתי הוא גם סיפק. שיטות ratiometric חד צבע נחשבות בדרך כלל עדיפה על אורך גל בודד או פרוטוקולים פסאודו-ratiometric כפול צבע, כפי שהם פחות רגישים להפרעות כגון הלבנה, להתמקד שינויים, וריאציות לייזר, ותיוג אחיד, המעוותים את האות הנמדדת. שיטה זו ניתן לשנות בקלות למדוד pH בסוגי תאי phagocytic אחרים, וzymosan יכול להיות מוחלף על ידי כל חלקיקים המכילים אמין אחרים, מחרוזי אינרטי למיקרואורגניזמים חיים. לבסוף, בשיטה זו ניתן להתאים לעשות שימוש בבדיקות ניאון אחרים רגישות לטווחי pH שונים או phagosom האחרפעילויות אל, מה שהופך את פרוטוקול כללי להדמיה התפקודית של phagosomes.

הם הבאים תוצאות נציג לניסוי שבו ה- pH phagosomal של נויטרופילים עכבר העיקריים מבודדים ממח העצם של פרא-סוג או Hvcn1 - / - עכברים הושוו. לניסוי מוצלח, חשוב להשיג מספיק phagosomes בתוך שדה הראייה בכל משך סרט הזמן לשגות, תוך הימנעות phagosomes רב מדי, שמאוחר יותר יהיה קשה יותר ?...

Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

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 ...

measuring-phagosome-ph-by-ratiometric-fluorescence-microscopy

Research

JoVE Journal

Immunology and Infection

13.8K Views.  University of Geneva. 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. 

Video: Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

מיקרוגל Assisted אבחון מהיר של מחל וירוס צמח על ידי מיקרוסקופי אלקטרוני הילוכים

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

ויזואליזציה של תגובות מושרות רעלן של חיידקים באמצעות מיקרוסקופ פלואורסצנטי תא החי

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

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 Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

מדידת תגובות פיזיולוגיות של<em&gt; תסיסנית</em&gt; עצב סנסורי כדי ליפידים פרומונים שימוש סידן הדמיה חיה

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

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 ...

"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.

&quot;Phagosome Closure Assay&quot; 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 ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

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 ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

להמחיש מוקש תאיים סחר על-ידי קרינה פלואורסצנטית שלמים הדמיה במיקרוסקופ

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

מדידת ודפורמביליות והטרוגניות תא אדום בדם על ידי Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

במבחנה מבנה הכלבי נויטרופילים מלכודת חוץ-תאית: דינמי וניתוח כמותי על-ידי קרינה פלואורסצנטית מיקרוסקופ

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...