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.).

بيان الأخلاق: تم تنفيذ جميع التلاعب الحيوانية بما يتفق تماما مع المبادئ التوجيهية للجنة البحوث الحيوانية من جامعة جنيف. 1. إعداد الأهداف أكلة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 20 ملغ من زيموزان المجففة إلى 10 مل الفوسفات العقيمة مخزنة المالحة (PBS). دوامة والحرارة في حمام الماء المغلي لمدة 10 دقيقة. بارد وأجهزة الطرد المركزي في 2000 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إزالة طاف، resuspend في 1 مل PBS ويصوتن لمدة 10 دقيقة في sonicator حمام الماء. نقل 500 ميكرولتر لاثنين من 1.5 مل أنابيب. أجهزة الطرد المركزي في 11000 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> تكرار غسل مع 500 ميكرولتر PBS. يمكن aliquoted في زيموزان المغلي، المجمدة وتخزينها في -20 ° C. </li><li style=";text-align:right;direction:rtl"> غسل زيموزان مرتين في 500 ميكرولتر الطازجة 0.1 M نا 2 CO 3، ودرجة الحموضة 9.3 على النحو الوارد أعلاه (الخطوة 1.2). </li><li style=";text-align:right;direction:rtl"> resuspend في 490 ميكرولتر بعد غسل النهائي. إضافة 10 ميكرولتر من 10 ملغ / مل المنحل FITC في ثنائي ميثيل sulfoxidه ([دمس])، دوامة، مع تغطية احباط، واحتضان مع الانفعالات لمدة 4 ساعة على RT. </li><li style=";text-align:right;direction:rtl"> لإزالة الصبغة غير منضم، وغسل النحو الوارد أعلاه (الخطوة 1.2)، لأول مرة مع 0.5 مل DMSO + 150 ميكرولتر PBS، ثم 0.5 مل DMSO + 300 ميكرولتر PBS، ثم 0.5 مل DMSO + 450 ميكرولتر PBS، ثم PBS وحده. </li><li style=";text-align:right;direction:rtl"> عد زيموزان باستخدام haemocytometer. إضافة 1 ميكرولتر من زيموزان إلى 500 ميكرولتر PBS، الدوامة، ثم إضافة 10 ميكرولتر إلى حافة الغرفة haemocytometer، حيث يلتقي ساترة وضعت على الشبكة المركزية. تحميل haemocytometer على المجهر مشرق الميدان مجهزة الهدف 20X. </li><li style=";text-align:right;direction:rtl"> التركيز على الزاوية العلوية اليسرى يحتوي على 16 الساحات والاعتماد جميع الجزيئات زيموزان في هذا المجال باستخدام عداد اليد رصيده. كرر مع الزاوية السفلى اليمنى. حساب تركيز زيموزان باستخدام المعادلة التالية: تركيز زيموزان = عدد / 2 × 500 × 10 4 زيموزان / مل. يمكن aliquoted زيموزان المسمى مثلجة وتخزينها في -20 ° C. </li><li style=";text-align:right;direction:rtl"> يستطهي واي زيموزان المسمىث أرنب الأجسام المضادة لمكافحة زيموزان في التخفيف 1: 100. دوامة واحتضان لمدة 1 ساعة في 37 ° C حمام الماء. يغسل مع 500 ميكرولتر PBS على النحو الوارد أعلاه (الخطوة 1.2). ملاحظة: يوصى صوتنة للغاية، خصوصا بعد طهاية، حيث يميل زيموزان لتشكيل كتل التي يمكن أن تجعل تحليل أكثر صعوبة في وقت لاحق. طهاية مع 65٪ ت / ت الفئران أو المصل البشري يمكن أن تستخدم كبديل. </li><li style=";text-align:right;direction:rtl"> عد زيموزان باستخدام haemocytometer كما هو موضح في الخطوات 1،7-1،8، وتمييع إلى 100 ​​× 10 6 الجسيمات / مل. طهاية مع التحفيز والتشجيع أكلة أخرى، مثل تكملة أو لا طهاية، قد يتم تنفيذ بدلا من ذلك. </li></ol> 2. لايف فيديو المجهر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عزل العدلات الماوس الأولية كما هو موضح بالتفصيل في مكان آخر 38. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بدلا من ذلك، استخدام أي نوع من الخلايا البلعمية. الحفاظ العدلات على الجليد في معهد روزويل بارك التذكاري (RPMI) 1640 متوسطة تستكمل مع 5٪ الجنين المصل في ربلة الساق (ت / ت)، 200 U البنسلين / streptomyciن، بتركيز 10 × 10 6 خلية / مل لتصبح جاهزة للاستخدام. قد يكون المصنف أنواع الخلايا الأخرى قبل 1-4 أيام. </li></ol></li><li style=";text-align:right;direction:rtl"> إضافة 2 × 10 5 (20 ميكرولتر) العدلات إلى وسط الزجاج السفلي 35 مم طبق، ونشر انخفاض بإضافة 50 ميكرولتر من المتوسطة واحتضان عند 37 درجة مئوية لمدة 5 دقائق للسماح للخلايا الالتزام. </li><li style=";text-align:right;direction:rtl"> إضافة الدفء 1 مل من محلول ملحي متوازن هانك (HBSS) إلى 37 درجة C الذي يحتوي على MPO محددة هيدرازيد المانع 4-أمينو (4 ABH، 10 ميكرومتر). مثبطات MPO غير محددة، مثل أزيد الصوديوم (5 ملم)، ويمكن أيضا أن تستخدم ولكن قد اقترحت مؤخرا لممارسة تأثيرات غير محددة إضافية على درجة الحموضة 39. </li><li style=";text-align:right;direction:rtl"> تركيب الطبق على المجهر widefield مضان مجهزة 37 ° C نظام التدفئة والهدف النفط 40X. احتضان الخلايا دون التصوير لمدة 10 دقيقة للسماح للخلايا ومعدات للتوازن. </li><li style=";text-align:right;direction:rtl"> ضبط إعدادات المجهر للحصول على نقل صورة مشرق الميدان [فاحد ذاتها أو على النقيض تدخل الفرق (DIC) إن وجدت] مع 440 نانومتر أو 490 نانومتر الإثارة و 535 نانومتر انبعاث كل 30 ثانية. </li><li style=";text-align:right;direction:rtl"> ضبط وقت التعرض وتردد الاستحواذ وفقا لنظام المجهر لتجنب الكثير من photobleaching من والضيائية واعتمادا على الأسئلة التجريبية التي طلب منها ذلك. بدء الحصول على الصور. </li><li style=";text-align:right;direction:rtl"> بعد 2 دقيقة، وإضافة 10 × 10 6 (50 ميكرولتر) opsonized FITC-زيموزان إلى وسط الطبق. ضبط الهدف: نسب الخلايا تبعا لنوع من بلعمية والهدف المستخدمة. </li><li style=";text-align:right;direction:rtl"> يستمر التصوير لمدة 30 دقيقة. بعد 30 دقيقة وقف اكتساب الوقت الفاصل بين وبدء الموقت. تحريك خشبة المسرح والتقاط 10 أو أكثر لقطات في مختلف المجالات من أجل صورة phagosomes الآخرين، كل ذلك ضمن 5 دقائق. </li></ol> 3. المعايرة ومراقبة التجارب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد الحلول درجة الحموضة المعايرة (موصوفة وصفات في الجدول رقم 1) قبل يوم من التجربة وتجميد في 10 مل قسامات.ذوبان الجليد الحلول المعايرة ودافئة إلى 37 درجة مئوية في حمام مائي. تحقق من درجة الحموضة مع متر الرقم الهيدروجيني وتسجيل الرقم الهيدروجيني الفعلية من الحلول. </li><li style=";text-align:right;direction:rtl"> جبل مضخة تحوي على الجزء السفلي صحن من الزجاج قبل الحصول على الصور وإجراء الفحص المجهري لقطات فيديو حية كما هو موضح أعلاه (القسم 2). </li><li style=";text-align:right;direction:rtl"> في نهاية فترة شراء 30 دقيقة تشغيل المضخة لإزالة الحل في طبق، إضافة 1 مل من محلول المعايرة الأول ووقف ضخ. </li><li style=";text-align:right;direction:rtl"> الانتظار حتى تستقر إشارة، عادة مدة 5 دقائق. أكرر مع كل حل المعايرة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أداء المعايرة واحدة على الأقل في اليوم التجريبي، ومعايرة بدءا من أعلى درجة الحموضة وبالتتابع عمل إلى أدنى مستوى، فضلا عن بدء من أدنى درجة الحموضة والعمل بشكل متتالي نحو الأعلى. </li></ol></li><li style=";text-align:right;direction:rtl"> كعنصر تحكم، إضافة 100 ميكرولتر من 100 ميكرومتر NADPH أوكسيديز المانع ثنائي الفينيل propionium يوديد (DPI) المخفف في HBSS إلى الخلايا في 900 ميكرولتر HBSS في طبق زجاجي القاع، واحتضان لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> بدء استحواذ وإضافة زيموزان على النحو الوارد أعلاه (الخطوة 2.7). بعد 15 دقيقة من البلعمة، إضافة 10 ميكرولتر من 10 ميكرومتر V-أتباز المانع concanamycin A (كونكا) المخفف في HBSS ويستمر التصوير لمدة 15 دقيقة إضافية. </li></ol> تحليل 4. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحليل الأفلام الوقت الفاصل بين واللقطات يبلوع. ملاحظة: الإرشادات التالية هي محددة ليماغيج. خطوة بخطوة قد تختلف الإرشادات على نطاق واسع اعتمادا على البرمجيات المستخدمة، ولكن هي وظائف الموضحة في هذا القسم متاح في معظم البرامج المهنية تحليل الصور. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فتح الصور مع البرامج المهنية تحليل الصور. طرح الخلفية في القناة 490 عن طريق رسم ROI مربع صغير مع مربع أداة التحديد المضلع في منطقة حيث لا توجد الخلايا، والنقر الإضافات&gt; BG الطرح من العائد على الاستثمار. ثم تحميل نفس ROI على قناة 440 بالنقر فوق تحرير&gt; اختيار&gt; استعادة التحديد وتكرار. </li><li style=";text-align:right;direction:rtl">جعل 32 بت نسبة صورة القناة 490 مقسوما على قناة 440 بالضغط العملية&gt; حاسبة صورة ...، واختيار الصور المناسبة في Image1 وو Image2 القوائم المنسدلة، ثم اختيار &#39;تقسيم&#39; في القائمة المنسدلة عملية ، والتحقق من 32 بت (تعويم) نتيجة الاختيار مربع. </li><li style=";text-align:right;direction:rtl"> تغيير نظرة متابعة الجدول الترميز اللوني لنسبة المتوافقة مع لون الترميز بالضغط صورة&gt; طاولات بحث&gt; قوس قزح RGB. عتبة الصورة نسبة إلى القضاء 0 واللانهاية بكسل بالضغط صورة&gt; ضبط&gt; العتبة .... ضبط أشرطة التمرير بحيث يظهر أن زيموزان باللون الأحمر وانقر فوق تطبيق، والتأكد من تحديد مجموعة بكسل خلفية خانة الاختيار نان ل. </li><li style=";text-align:right;direction:rtl"> الجمع بين هذه النسبة كومة مع قناة مشرق الميدان في مركب متعدد القنوات بالضغط صورة&gt; اللون&gt; دمج القنوات، واختيار الصورة مشرق الميدان في القائمة قناة المنسدلة الرمادي وصورة نسبة في القائمة المنسدلة قناة خضراء، و اختيارإبقاء مصدر الصور فحص مربع. مسح الأفلام مرور الزمن أو لقطات لتحديد أي phagosomes هم لتحليلها. قناة مشرق الميدان مفيدة لهذا الغرض. </li><li style=";text-align:right;direction:rtl"> رسم مناطق الفائدة (ROI) باستخدام أداة التحديد البيضاوي المضلع حول phagosomes، وإضافتها إلى مدير ROI بالضغط تحليل&gt; أدوات&gt; مدير ROI&gt; إضافة. قياس نسبة كثافة زيموزان من خلال النقر المزيد&gt; متعددة للقياس واجتثاث اختيار صف واحد لكل شريحة الاختيار مربع. </li><li style=";text-align:right;direction:rtl"> للأفلام مرور الزمن، المسار phagosomes في وقت واحد من خلال الاعتماد على العائد على الاستثمار، وكتابة على Ctrl + M لقياس كل نقطة زمنية، وتحريك ROI عند الضرورة لتتبع القيم شدة مع مرور الوقت. نسخ القياسات من نافذة نتائج في برنامج جداول البيانات. ملاحظة: تحليل 15 (5 دوام دورات في التجربة × 3) تجارب مستقلة غير مثالية، ولكن أكثر أو أقل قد تكون هناك حاجة تبعا لتقلب الملاحظة. إنقاذ رويس ينصح دائما. بعض حزم البرمجيات تسمح ريجي صورةstration والتحديث الحيوي رويس، وتسهيل هذا الجزء من التحليل. </li></ol></li><li style=";text-align:right;direction:rtl"> التحويل من مضان للقيم الرقم الهيدروجيني <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قياس نسبة 490/440 لphagosomes في معايرة الأفلام مرور الزمن كما هو موضح في القسم 4.1. </li><li style=";text-align:right;direction:rtl"> في جدول بيانات، رسم القيم نسبة مع مرور الوقت، وحساب متوسط ​​قيم نسبة من كل phagosomes داخل مجال الرؤية (عادة ما بين 5-20) من 5 نقاط وقت خلال منتصف الفترة الزمنية المقابلة لكل درجة الحموضة المعايرة حل. (انظر أيضا مربعات حمراء في الشكل 5A). </li><li style=";text-align:right;direction:rtl"> رسم هذه 490/440 متوسط ​​قيم نسبة الرقم الهيدروجيني ضد المقاسة الفعلية. الجمع بين ما لا يقل عن 3 المعايرة مستقلة في رسم بياني واحد. استخدام حزمة البرامج الإحصائية أو العددية لإجراء الانحدار غير الخطية (أفضل منحنى نوبة)، وعادة لمعادلة السيني. ملاحظة: على سبيل المثال، في الشكل 5B كانت المعادلة المستخدمة لالسيني بولتزمان [Y = أسفل + ((من أعلى إلى أسفل)/ (1 + EXP ((V50-X) / المنحدر))]، حيث قيم Y هي نسب 490/440، X هي قيم الرقم الهيدروجيني وأعلى، أسفل، وتحسب V50 والمنحدر المعلمات من قبل البرنامج. </li><li style=";text-align:right;direction:rtl"> استخدام المعادلة التي تم الحصول عليها لتحويل القيم نسبة لدرجة الحموضة. ملاحظة: على سبيل المثال، في الشكل 5B المعادلة التي حصل عليها لمنحنى المعايرة بحضور أزيد الصوديوم 490/440 نسبة = 1.103 + [(2،89-1،103) / (1 ​​+ EXP ((6.993 درجة الحموضة) /0.9291)] . حل لدرجة الحموضة يعطي المعادلة التالية: الرقم الهيدروجيني = 6،993-0،9291 * [LN ((1.178 / (نسبة 1.103)) - 1))]. </li></ol></li></ol>

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C., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+phagosomal+pH+in+neutrophils.">Determinants of the phagosomal pH in neutrophils.</a> J Biol Chem. 277 (8), 6059-6066 (2002).</li><li>Segal, A. W., Geisow, M., Garcia, R., Harper, A., Miller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+burst+of+phagocytic+cells+is+associated+with+a+rise+in+vacuolar+pH.">The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.</a> Nature. 290 (5805), 406-409 (1981).</li><li>Savina, 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=NOX2+controls+phagosomal+pH+to+regulate+antigen+processing+during+crosspresentation+by+dendritic+cells.">NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells.</a> Cell. 126 (1), 205-218 (2006).</li><li>Rybicka, J. M., Balce, D. R., Chaudhuri, S., Allan, E. R., Yates, R. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Interferon-inducible+lysosomal+thiol+reductase+(GILT)+maintains+phagosomal+proteolysis+in+alternatively+activated+macrophages.">gamma-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.</a> J Biol Chem. 289 (46), 31891-31904 (2014).</li><li>Sanjurjo, L., 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=The+scavenger+protein+apoptosis+inhibitor+of+macrophages+(AIM)+potentiates+the+antimicrobial+response+against+Mycobacterium+tuberculosis+by+enhancing+autophagy.">The scavenger protein apoptosis inhibitor of macrophages (AIM) potentiates the antimicrobial response against Mycobacterium tuberculosis by enhancing autophagy.</a> PLoS One. 8 (11), e79670 (2013).</li><li>Yuan, Z., Zhi, L., Li-Juan, Z., Hong-Tao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utility+of+Chloroquine+in+Cancer+Therapy.">The Utility of Chloroquine in Cancer Therapy.</a> Curr Med Res Opin. , 1-12 (2015).</li><li>Ohkuma, S., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+measurement+of+the+intralysosomal+pH+in+living+cells+and+the+perturbation+of+pH+by+various+agents.">Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.</a> Proc Natl Acad Sci U S A. 75 (7), 3327-3331 (1978).</li><li>El Chemaly, 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=VSOP/Hv1+proton+channels+sustain+calcium+entry,+neutrophil+migration,+and+superoxide+production+by+limiting+cell+depolarization+and+acidification.">VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.</a> J Exp Med. 207 (1), 129-139 (2010).</li><li>Levine, A. 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The authors have nothing to disclose.

على الرغم من أن أكثر استهلاكا للوقت من الطرق البديلة، مثل التحليل الطيفي وFACS، التي تستخدم استراتيجية مماثلة باستخدام صبغة حساسة درجة الحموضة بالإضافة إلى أهداف ولكن قياس متوسط ​​درجة الحموضة من سكان phagosomes و الفحص المجهري ويقدم العديد من المزايا. الأول هو أن الداخل...

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

البلعمة هي عملية أساسية من خلالها الخلايا المناعية الفطرية تبتلع البكتيريا والخلايا أفكارك أو الجسيمات الأجنبية الأخرى من أجل قتل أو تحييد المواد المبتلعة، أو تقديمه كمستضدات والشروع في الاستجابات المناعية التكيفية. الرقم الهيدروجيني للphagosomes معلمة الحرجة التي تنظم الانشطار أو الاندماج مع endomembranes وتفعيل الانزيمات المحللة للبروتين، والأحداث التي تسمح للفجوة أكلة إلى أن تنضج في عضية الهادمة. بالإضافة إلى ذلك، هناك حاجة النبات من H + لإنتاج مستويات عالية من أنواع الاكسجين التفاعلية (ROS)، التي تعتبر ضرورية لقتل كفاءة ويشير إلى الأنسجة المضيفة الأخرى. العديد من مسببات الأمراض داخل الخلايا البلعمية تخريب قتل عن طريق الحد من phagosomal تحمض، وتسليط الضوء على أهمية درجة الحموضة في يبلوع علم الأحياء. نحن هنا تصف طريقة ratiometric لقياس درجة الحموضة phagosomal في العدلات باستخدام ثيوسيانات فلوريسئين (FITC) -labeled زيموزان كما أكلة TARGخدمات الاختبارات التربوية، والتصوير الخلية الحية. ويستند هذا الفحص على خصائص مضان FITC، التي تطفئ بواسطة الرقم الهيدروجيني الحمضية عندما متحمس في 490 نانومتر ولكن ليس عندما متحمس في 440 نانومتر، مما يتيح التقدير الكمي لنسبة تعتمد على درجة الحموضة، بدلا من مضان المطلق، من صبغة واحدة. وتقدم أيضا بروتوكول مفصلة لأداء الموقع صبغ المعايرة وتحويل النسبة إلى قيم الرقم الهيدروجيني حقيقية في. تعتبر الطرق ratiometric واحد صبغ عموما متفوقة على الطول الموجي واحد أو بروتوكولات الزائفة ratiometric ثنائي الصبغة، لأنها أقل حساسية للاضطرابات مثل تبيض، والتركيز التغييرات، والاختلافات الليزر، ووضع العلامات متفاوتا، التي تشوه الإشارة المقاسة. هذه الطريقة يمكن تعديلها بسهولة لقياس درجة الحموضة في غيرها من أنواع الخلايا البلعمية، وزيموزان يمكن الاستعاضة عن أي الجسيمات التي تحتوي على الأمينات الأخرى، من حبات الخاملة إلى الكائنات الحية المجهرية. وأخيرا، وهذه الطريقة يمكن تكييفها للاستفادة من تحقيقات الفلورسنت أخرى حساسة لدرجة الحموضة نطاقات مختلفة أو phagosom الآخرأنشطة القاعدة ويجعل من بروتوكول المعمم للتصوير وظيفي من phagosomes.

وفيما يلي نتائج ممثل للتجربة حيث درجة الحموضة phagosomal العدلات الماوس الابتدائية معزولة عن نخاع العظام من النوع البري أو Hvcn1 - / - وتمت مقارنة الفئران. لتجربة ناجحة، فمن المهم الحصول على ما يكفي من phagosomes داخل مجال الرؤية خلال فترة كاملة من الفيلم الوقت ال?...

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.

قياس درجة الحموضة يبلوع التي كتبها Ratiometric المجهر مضان

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.

الميكروويف بمساعدة التشخيص السريع للأمراض النباتية من الفيروسات المجهر الإلكتروني ناقل

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.

تصور الردود السم البكتيري المستحثة عن طريق لايف مضان المجهري الخليوي

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.

دراسة يحلول يبلوعي النشوء الأحيائي في الضامة لايف

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.

قياس الاستجابات الفسيولوجية لل<em&gt; ذبابة الفاكهة</em&gt; الخلايا العصبية الحسية إلى الدهن الفيرومونات عن طريق لايف التصوير الكالسيوم

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.

التصوير Ratiometric الرقم الهيدروجيني خارج الخلية في الأغشية الحيوية الأسنان

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;يبلوع إغلاق الفحص&quot; لتصور يبلوع تشكيل في ثلاثة أبعاد عن طريق التأمل إجمالي الداخلية نيون الميكروسكوب (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.

تصوير العدلات يبلوع والسيتوبلازم باستخدام الرقم الهيدروجيني المؤشر 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.

تصور داخل الخلايا المطوقة الاتجار بواسطة عمر Fluorescence التصوير المجهري

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.

قياس ديفورمابيليتي وعدم تجانس الخلايا الحمراء في الدم من اكتاسيتوميتري

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.

في المختبر تشكيل الكلاب العَدلات فخ خارج الخلية: التحليل الكمي ودينامية بالفحص المجهري الأسفار

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