We thank Dr. Gilles Dupuis for critical reading of the manuscript. 
Funding
This work was supported by the Canadian Institutes for Health Research, grant # MOP-126173. C.M.D is a member of the Fonds de la Recherche en Sant&#233; (FRSQ)-funded Centre de Recherche Clinique &#201;tienne LeBel.

1. הכנה של פתרונות לסלולריים pH כיול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף את התרכובות הבאות לחמישה צינורות נפרדים 50 מיליליטר חרוטי להכין פתרונות 5 לשמש לכיול: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> NaCl (1 מיליליטר של 1 M NaCl) (1 M NaCl = 0.58 גרם ב10 H מיליליטר 2 0) </li><li style=";text-align:right;direction:rtl"> KCl (6.75 מיליליטר של 1 M KCl) (1 M KCl = 0.75 גרם ב10 H מיליליטר 2 0) </li><li style=";text-align:right;direction:rtl"> גלוקוז (1 מיליליטר של 1 M D-גלוקוז) (1M D-גלוקוז = 1.80 גרם ב10 H מיליליטר 2 0) </li><li style=";text-align:right;direction:rtl"> MgS0 4 (0.05 מיליליטר של 1 M MgSO 4) (1 M MgSO 4 = 1.20 גרם ב10 H מיליליטר 2 0) </li><li style=";text-align:right;direction:rtl"> 20 HEPES מ&quot;מ (1 מיליליטר של 1M HEPES) (1 M HEPES = 2.38 גרם ב10 H מיליליטר 2 0) </li><li style=";text-align:right;direction:rtl"> CaCl 2 (0.05 מיליליטר של 1M CaCl 2) (ז גרם CaCl 2 = 1.47 1 ב10 H מיליליטר 2 0) הכן את כל הפתרונות במזוקקים H 2 O (DDH 2 O) הכפול. </li></ul></li><li style=";text-align:right;direction:rtl"> להשלים כל פתרון עם 35 מיליליטר של DDH 2 O ולערבב עם בוחש מגנטי עד כהlution מושגת. </li><li style=";text-align:right;direction:rtl"> התאם את ה-pH של כל פתרון עם 1 N KOH או 1 N HCl להשיג ערכי ה-pH של 5.5, 6.0, 6.5, 7.0 ו7.5. לאחר מכן, להתאים את עוצמת הקול עם מים deionized נפח סופי של 50 מיליליטר. </li><li style=";text-align:right;direction:rtl"> מדד 5 מ&quot;ג של nigericin ולהוסיף אותו ל670 μl של EtOH המוחלט להכין 10 מ&quot;מ nigericin פתרון מניות. זהירות: nigericin הוא רעיל מאוד ויש לטפל בם בזהירות רבה (כפפות, משקפי מגן ומסכה). מערבבים את הפתרון על ידי היפוך עד nigericin הוא נמס לגמרי. </li><li style=";text-align:right;direction:rtl"> הוספת 0.05 מיליליטר של 10 מ&quot;מ nigericin (ריכוז סופי 10 מיקרומטר) לכל פתרון כיול. Nigericin הוא ionophore שמגביר את חדירות קרום תא וגורם לאיזון של רמת חומציות תוך תאית וחוץ תאי בנוכחות ריכוז depolarizing של תאי K +. </li><li style=";text-align:right;direction:rtl"> ניתן לאחסן את פתרונות כיול לחודש 1 על 4 מעלות צלזיוס </li></ol> 2. הכנת תא <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תחת safet ביולוגימכסה המנוע y, תאי זרע ב35 מ&quot;מ מנות תרבות x 10 מ&quot;מ 2 מיליליטר של מדיום גידול בתוספת 10% בסרום שור עוברי ואנטיביוטיקה. השתמש בכמות התאים הדרושה כדי להבטיח מפגש 50 כ% לאחר 24 שעות. הערה: בניסויים שלנו, אנו משתמשים בקו HT-1080 התא וצלחת 1 x 10 5 תאים לכל צלחת תרבות. </li><li style=";text-align:right;direction:rtl"> לקבוע את מספר מנות התרבות הדרושה לניסוי וצלחת 5 מנות תרבות נוספות שמשמש לכיול. </li><li style=";text-align:right;direction:rtl"> מנות תרבות המקום בC / 5% CO 2 באינקובטור 37 מעלות לפחות 24 שעות. </li></ol> 3. הכנת בדיקות pH חישה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן פתרון מניות 1M של חללית ה-pH ניאון 8-hydroxypyrene-1, 3,6 חומצת trisulfonic, מלח trisodium (HPTS / pyranine) בDDH 2 0. פתרון זה חייב להיות מאוחסן ב RT ומוגן מפני אור. </li><li style=";text-align:right;direction:rtl"> הכן פתרון מניות של 1 מ&quot;מ 5 - (ו- 6)-carboxyl seminaphthorhodafluor acetoxymethyl אסטר אצטט (C-snarf-1 בבוקר, נקרא thereaחרי snarf-1) על ידי המסת 1 מ&quot;ג של snarf-1 ב1.76 מיליליטר של DMSO. לערבב ביסודיות על ידי pipetting חוזר ונשנה. פתרון snarf-1 חייב להיות מאוחסן ב-20 מעלות צלזיוס ומוגנת מפני אור. </li></ol> 4. תיוג תא <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שישה עשר שעות לפני ההדמיה לחיות תאים, להוסיף 2 μl של פתרון מניות 1M HPTS (ריכוז סופי 1 מ&quot;מ) לכל מנה תרבות המכילה 2 מיליליטר של מדיום שלם. </li><li style=";text-align:right;direction:rtl"> להחזיר את הכלים התרבות לחממה. הבדיקה HPTS היא תא impermeant, ולכן לתווית האברונים הבדיקה חייבת להיות נלקחת על ידי pinocytosis. מצב זה מחייב את incubations תא עם HPTS מבוצע במדיום תרבות, בתוספת סרום. </li><li style=";text-align:right;direction:rtl"> לאחר דגירה עם HPTS, לשטוף את התאים פעמיים עם PBS סטרילי ולהוסיף 2 מיליליטר של מדיום סרום ללא לפחות 2 שעות. דגירה עם snarf-1 צריכה להתבצע במדיום סרום ללא בגלל סרום עלול למנוע הספיגה של החללית. </li><li style=";text-align:right;direction:rtl"> הוסף 10 μl של פתרון מניות snarf-1 1 מ&quot;מ להשיג afריכוז inal של 5 מיקרומטר ולאפשר לתאי דגירה של 20 דקות על 37 מעלות צלזיוס, באור עמום. ריכוז C-snarf-1 וזמן דגירה יכולים להשתנות בהתאם לסוג תא. </li><li style=";text-align:right;direction:rtl"> בסוף הדגירה, לשטוף תאי פעמים PBS סטרילי ולהוסיף 2 מיליליטר של מדיום ללא סרום. </li></ol> 5. תוספת של מאפנני pH <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> על מנת להעריך האם השיטה יכולה לזהות שינויי pH בתאים, חומרים כימיים שונים המווסתים pH תאיים ניתן להשתמש: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Bafilomycin A1, מעכב ספציפי של V-ATPase 5 </li><li style=";text-align:right;direction:rtl"> איזופורם מחליף Na + / H + 1 (NHE-1) 6 </li><li style=";text-align:right;direction:rtl"> NH 4 Cl, בסיס חלש, שמגביר את רמת חומציות תוך תאית 7 </li></ul><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן פתרונות מניות של חומרים כימיים שהוזכרו לעיל: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Bafilomycin A1: להוסיף 10 מיקרוגרם של bafilomycin A1 ל160 μl של DMSO להשיג פתרון מניות של 100 מיקרומטר </li><li style=";text-align:right;direction:rtl"> EIPA: להוסיף 25 מ&quot;ג EIPA ל1.67 מיליליטר של מתנול לobtaiריכוז סופי na של 50 מ&quot;מ </li><li style=";text-align:right;direction:rtl"> NH 4 Cl: לפזר 0.53 גרם של אמוניום כלוריד ב10 מיליליטר של DDH 2 O להשיג ריכוז סופי של 1 מ&quot;מ. </li></ul> הערה: כל פתרונות המניות חייבים להישמר על 4 מעלות צלזיוס למעט A1 bafilomycin אשר חייב להיות מאוחסן ב-20 מעלות צלזיוס ומוגנות מפני אור. </li><li style=";text-align:right;direction:rtl"> בתרבות מנות נפרדות, מוסיף אחד מהחומרים הכימיים כמפורט להלן: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 2 μl של 100 bafilomycin מיקרומטר A1 (ריכוז סופי 100 ננומטר) </li><li style=";text-align:right;direction:rtl"> 1 μl של 50 מ&quot;מ EIPA (מיקרומטר ריכוז 25 הסופי) </li><li style=";text-align:right;direction:rtl"> 40 μl של 1M 4 Cl NH (ריכוז סופי 20 מ&quot;מ) </li></ul></li><li style=";text-align:right;direction:rtl"> דגירה התאים למשך 30 דקות ב37 ° C. </li></ol></li><li style=";text-align:right;direction:rtl"> המשך לרכישת תמונה. </li></ol> 6. מפרט של מיקרוסקופ Live-תא ההדמיה והגדרות רכישה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבצע הדמיה לחיות תאים באמצעות מטרת 40X רכוב על microsco סריקה הפוכה רפאים confocalpe מצויד בשלב XY ממונע, חדר שיכול להכיל מנות תרבות תחתית זכוכית בחממת במה (טמפרטורה, לחות, CO 2) מבוקרת לסביבה. </li><li style=";text-align:right;direction:rtl"> מכשיר confocal (אולימפוס FV1000) משמש כאן מצויד ב: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ננומטר 405 לייזר דיודה </li><li style=";text-align:right;direction:rtl"> לייזר ארגון 40 mW (458 ננומטר, 488 ננומטר, 515 ננומטר) </li><li style=";text-align:right;direction:rtl"> לייזר ניאון הליום (543 ננומטר) </li><li style=";text-align:right;direction:rtl"> מטרת פלואוריט 10X, NA0.4 </li><li style=";text-align:right;direction:rtl"> מטרת LUCPLFLN 40X, NA0.60 </li><li style=";text-align:right;direction:rtl"> מטרת שמן PLAPONSC תכנית APO 60x, NA1.4 </li><li style=";text-align:right;direction:rtl"> DM מראה dichroic 405/458/488/543 </li><li style=";text-align:right;direction:rtl"> SDM 560 וSDM 640 מסננים dichroic </li><li style=";text-align:right;direction:rtl"> מסנן מחסום BA655-755 </li><li style=";text-align:right;direction:rtl"> BS20/80 מפצל קרן </li><li style=";text-align:right;direction:rtl"> תוכנה לניתוח נתונים לרכישת תמונה וניתוח, </li></ul></li><li style=";text-align:right;direction:rtl"> ברגע חממת השלב התחממה ל37 מעלות צלזיוס ומיקרוסקופ מוגדר, מניח צלחת תרבות מודגרות בעבר עם HPTS וsnarf-1 לחדר ההדמיה שלב confocal. </li><li style=";text-align:right;direction:rtl">חרי איתור שטח של עניין, התאם את הגדרות רכישת תוכנת איור 1. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדר את שני סטי ערוץ וירטואליים. </li><li style=";text-align:right;direction:rtl"> ליזום את השלב הראשון שמתאים לעירור HPTS ב405 ננומטר ואוסף פליטה ב 505-525 ננומטר ועירור snarf-1 ב543 ננומטר ואוסף פליטה ב 570-600 ננומטר. </li><li style=";text-align:right;direction:rtl"> ליזום את השלב השני שמתאים לעירור HPTS ב458 ננומטר ואוסף פליטה ב 505-525 ננומטר ועירור snarf-1 ב543 ננומטר ואוסף פליטה ב 640-650 ננומטר. </li><li style=";text-align:right;direction:rtl"> צמצם confocal מוגדר 175 מיקרומטר כברירת מחדל וצמצם אופטימלי צריך להיות מוגדר באופן ידני ב300 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> קח את התמונה על ידי ערמת חלקים סידוריים אופקיים אופטיים (800 800 פיקסלים) של 0.4 מיקרומטר עובי. </li></ul></li></ol> הערה: מטרת 40X היא שימושית כדי לבחון כמה תאים בו זמנית ועובדת היטב לצפייה אברון. עם זאת, ברזולוציה מרחבית ועוצמת אות הן בטr המושג באמצעות אובייקטיבי שמן 60x. בנוסף, אין שום עירור או פליטת הצטלבות בין שני הצבעים. 7. כיול של בדיקות pH חישה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני הכיול, להסיר בינוני ממנת התרבות מודגרות בעבר עם HPTS וsnarf-1, לשטוף פעמיים עם PBS סטרילי ולאחר מכן להוסיף 2 מיליליטר של אחד מפתרוני הכיול חמש. </li><li style=";text-align:right;direction:rtl"> מניחים את צלחת התרבות לתוך תא שלב confocal. </li><li style=";text-align:right;direction:rtl"> המשך לרכישת הזמן לשגות תמונה בהתאם להגדרות הבאות: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדר את מספר התמונות ופעמים משך מרווח לניסוי. הערה: אנו משתמשים בדרך כלל מרווח 1 דקות בין תמונות עד 30 דקות. </li><li style=";text-align:right;direction:rtl"> לתכנת את התריס כדי לסגור בין הרכישה של כל תמונה. </li></ol></li><li style=";text-align:right;direction:rtl"> רשום את עוצמת הקרינה לדקות בתוכנת גיליון אלקטרוני ולקבוע את הזמן שבו עוצמת הקרינה היא יציבה. ה-pH תאיים היא בדרך כלל יציבה לאחר 15-20 דקות. </li><li style=";text-align:right;direction:rtl"> REPEAT צעדים 7.1-7.4 באמצעות צלחת תרבות חדשה עבור כל פתרון כיול הנותר. </li></ol> 8. רכישת נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ברגע שהכיול נעשה, לרכוש נתונים לדוגמה 4-5 שדות שנבחרו באקראי המכילים 15-20 תאים לכל תחום. שתי תמונות מתקבלות עבור כל שדה: אחד לתיוג HPTS (שלפוחית) והשני עבור תיוג snarf-1 (cytosol). </li><li style=";text-align:right;direction:rtl"> הקרינה רקע (אזור תאי) היא הופחתה באופן דיגיטלי מתמונות ניאון של שני הבדיקות. שלפוחית ​​שהכותרת וcytosol מהתמונות המאוחסנות שלהם נבחרו באמצעות מסיכה בינארי. עבור כל בדיקה pH חישה, עוצמות הקרינה נמדדות בקנה מידה מספרי (0-4,095 קנה מידה ברמה אפורה). עוצמה ממוצעת של כל הפיקסלים באברון או ציטופלסמה מחושבת וערכים משמשים לחישוב יחס הקרינה. </li></ol> 9. ניתוח נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חבילות תוכנה שונות זמינות באופן מסחרי לניתוח נתונים. כלומרתוכנת FluoView. </li><li style=";text-align:right;direction:rtl"> בגיליון אלקטרוני, ערכי שיא ולחשב יחס הקרינה באופן הבא: במקרה של HPTS, R = (F 458 ננומטר / F 405 ננומטר), ובמקרה של snarf-1, R = (ננומטר F 644 ננומטר / F 584 ). </li><li style=";text-align:right;direction:rtl"> להתוות את הנתונים באמצעות תוכנת גרפים מדעית כגון GraphPad פריזמה. העלילה צריכה לשרטט יחסי הקרינה (ציר ה-Y) כפונקציה של ה-pH (ציר ה-X). התאמת עקומות מושגת באמצעות רגרסיה שאינה ליניארי. </li><li style=";text-align:right;direction:rtl"> לאסוף עוצמות הקרינה של הדגימות כמתואר בשלב 8.2 ולחשב יחס הקרינה. השתמש עקומות כיול להפוך יחסים לערכי ה-pH. </li></ol>

<ol>
	<li>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=pH+Homeostasis+of+cellular+organelles.">pH Homeostasis of cellular organelles.</a> News Physiol Sci. 17, 1-5 (2002).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nat Rev Cancer. 11, 671-677 (2011).</li><li>Hinton, A., 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=Function+of+a+subunit+isoforms+of+the+V-ATPase+in+pH+homeostasis+and+in+vitro+invasion+of+MDA-MB231+human+breast+cancer+cells.">Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells.</a> J Biol Chem. 284, 16400-16408 (2009).</li><li>Overly, C. C., Lee, K. D., Berthiaume, E., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+intraorganelle+pH+in+the+endosomal-lysosomal+pathway+in+neurons+by+using+ratiometric+imaging+with+pyranine.">Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine.</a> Proc Natl Acad Sci U S A. 92, 3156-3160 (1995).</li><li>Drose, S., Altendorf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycins+and+concanamycins+as+inhibitors+of+V-ATPases+and+P-ATPases.">Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases.</a> J Exp Biol. 200, 1-8 (1997).</li><li>Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., Cala, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NHE1+inhibition+by+amiloride-+and+benzoylguanidine-type+compounds.+Inhibitor+binding+loci+deduced+from+chimeras+of+NHE1+homologues+with+endogenous+differences+in+inhibitor+sensitivity.">NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity.</a> J Biol Chem. 282, 19716-19727 (2007).</li><li>Alfonso, A., Cabado, A. G., Vieytes, M. R., Botana, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-pH+crosstalks+in+rat+mast+cells:+cytosolic+alkalinization,+but+not+intracellular+calcium+release,+is+a+sufficient+signal+for+degranulation.">Calcium-pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation.</a> Br J Pharmacol. 130, 1809-1816 (2000).</li><li>Han, J., Burgess, K. <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+for+intracellular+pH.">Fluorescent indicators for intracellular pH.</a> Chem Rev. 110, 2709-2728 (2010).</li><li>Giuliano, K. A., 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=Determination+of+intracellular+pH+of+BALB/c-3T3+cells+using+the+fluorescence+of+pyranine.">Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine.</a> Anal Biochem. 167, 362-371 (1987).</li><li>Willoughby, D., Thomas, R., Schwiening, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+intracellular+pH+changes+on+resting+cytosolic+calcium+in+voltage-clamped+snail+neurones.">The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones.</a> J Physiol. 530, 405-416 (2001).</li></ol>

No conflict of interest declared.

יש צורך במדידת pH כמותי בתחום ההדמיה לחיות תאים של תאים סלולריים שונים כדי למדוד במדויק את שינויי ה-pH בתגובות תא לאתגרים חיצוניים. עם זאת, אחד המכשולים העיקריים שנותר הוא בקלות ובאופן ספציפי כדי למקד את התאים סלולריים. מבחינה זו, מספר מחקרים הדגישו את השימוש בHPTS כאינדי...

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Solutions for calibration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">D-Glucose (dextrose)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">D16-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Magnesium sulfate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M65-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">HEPES</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Calcium chloride dihydrate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-1612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Nigericin</td>
			<td style="width:136px;">Calbiochem</td>
			<td align="right" style="width:119px;">481990</td>
			<td>Toxic by inhalation or in contact with skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;">Keep protected from the light at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Culture dishes &#8709; 35mm</td>
			<td style="width:136px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH-sensing probes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">H-348</td>
			<td>Keep away from the light at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">5-(and-6)-Carboxy SNARF&#174;-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">C-1271</td>
			<td>Keep away from the light and humidity at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Dimethyl Sufoxide (DMSO)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">BP231-1&#160;</td>
			<td>Harmful</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium phosphate monobasic</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">S369-1</td>
			<td>115 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium phosphate monobasic</td>
			<td style="width:136px;">J.T Baker</td>
			<td style="width:119px;">3246-01</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Autoclave and adjust pH to 7.4</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 1X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">PBS 100X</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>50 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td>40,5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deoinized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH modulators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">&#160;Bafilomycin A1</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">B-1793</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Ammonium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">A649-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous ph measurement in endocytic and cytosolic compartments in living cells using confocal microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

למדידה בו זמנית של ה-pH בשני תאית ותאי תאי endosomal / lysosomal, בדיקות ratiometric ניאון pH חישת snarf-1 וHPTS היו בשימוש. Snarf-1 מוגבל לתא cytosolic אילו HPTS מאפשר מדידת pH ratiometric של תא endosomal / lysosomal. תא טעינה עם HPTS ל16 שעות מאפשרת הלוקליזציה סלקטיבית שלה בendosomes / lysosomes 4. דוגמא אופיינית של הקרינה נ...

Watch this Scientific Journal Video about Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy at JoVE.com

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

מדידת pH בו זמנית בendocytic ותאי Cytosolic בתאים חיים באמצעות מיקרוסקופיה confocal

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

simultaneous-ph-measurement-endocytic-cytosolic-compartments-living

Research

JoVE Journal

Biology

14.9K Views.  Université de Sherbrooke. Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells. 

Video: Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

צורה הדמיה תא שינוי חיים תסיסנית עוברים

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

הקרינה מבוסס מדידה הכניסה חנות המופעל הסידן בתאים חיים: מתא הסרטן תרבותי כדי סיב שריר שלד

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

מדידה של pH vacuolar וCytosolic<em&gt; In Vivo</em&gt; בהשעיות תא שמרים

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

ניתוח Craniofacial המורפוגנזה בדג הזברה שימוש 4D מיקרוסקופיה confocal

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

במשובט מעקב vivo של גזע hematopoietic ובתאים המסומנים על ידי חמישה חלבונים ניאון באמצעות Confocal וmultiphoton מיקרוסקופית

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

מדידות סידן cytosolic בתאי האפיתל כליות על ידי cytometry הזרימה

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

זמן לשגות הדמיה ברזולוציה גבוהה וניתוח אוטומטי של Dynamics microtubule In Living האדם טבורי וריד לתאי אנדותל

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

מדידה בו זמנית של סידן מיטוכונדריאלי מיטוכונדריאלי ממברנה פוטנציאלית בתאים חיים באמצעות פלורסנט מיקרוסקופית

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

ממשק משתמש גרפי למעקב בסיוע תוכנה של ריכוז חלבון בהפרעות ניידות דינמיות

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

חיטוט mRNA קינטיקה בחלל וזמן ב Escherichia קולי באמצעות שני צבעים פלואורסצנטיות מולקולה אחת ב סיטו הכלאה

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...