فإن الكتاب أود أن أشكر دايل Hirschkorn من معهد بحوث النظم الدم لمساعدته مع تدفق عداد الكريات ضبط الصك. كان مدعوما من المعاهد الوطنية للصحة هذا العمل يمنح HL095470 وU01 HL072268 وعقود وزارة الدفاع W81XWH-10-1-0023 وW81XWH-2-0028.

<ol>
	<li>Andaloussi, S., M&#228;ger, I., Breakefield, X. O., Wood, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+biology+and+emerging+therapeutic+opportunities.">Extracellular vesicles: biology and emerging therapeutic opportunities.</a> Nature Reviews Drug Discovery. 12 (5), 347-357 (2013).</li><li>Sugawara, A., Nollet, K. E., Yajima, K., Saito, S., Ohto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+platelet-derived+microparticle+formation--and+possible+side+effects-with+prestorage+leukofiltration+of+whole+blood.">Preventing platelet-derived microparticle formation--and possible side effects-with prestorage leukofiltration of whole blood.</a> Archives of Pathology &amp; Laboratory Medicine. 134 (5), 771-775 (2010).</li><li>Th&#233;ry, C., Ostrowski, M., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+as+conveyors+of+immune+responses.">Membrane vesicles as conveyors of immune responses.</a> Nature Reviews Immunology. 9 (8), 581-593 (2009).</li><li>Mause, S. F., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles+Protagonists+of+a+Novel+Communication+Network+for+Intercellular+Information+Exchange.">Microparticles Protagonists of a Novel Communication Network for Intercellular Information Exchange.</a> Circulation Research. 107 (9), 1047-1057 (2010).</li><li>Bouvy, C., Gheldof, D., Chatelain, C., Mullier, F., Dogne, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributing+role+of+extracellular+vesicles+on+vascular+endothelium+haemostatic+balance+in+cancer.">Contributing role of extracellular vesicles on vascular endothelium haemostatic balance in cancer.</a> Journal of Extracellular Vesicles. 3, (2014).</li><li>Hood, J. L., San, R. S., Wickline, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+Released+by+Melanoma+Cells+Prepare+Sentinel+Lymph+Nodes+for+Tumor+Metastasis.">Exosomes Released by Melanoma Cells Prepare Sentinel Lymph Nodes for Tumor Metastasis.</a> Cancer Research. 71 (11), 3792-3801 (2011).</li><li>Gatti, S., Bruno, S., 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=Microvesicles+derived+from+human+adult+mesenchymal+stem+cells+protect+against+ischaemia-reperfusion-induced+acute+and+chronic+kidney+injury.">Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury.</a> Nephrology Dialysis Transplantation. 26 (5), 1474-1483 (2011).</li><li>Barteneva, N. S., Fasler-Kan, E., 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=Circulating+microparticles:+square+the+circle.">Circulating microparticles: square the circle.</a> BMC Cell Biology. 14 (1), 23 (2013).</li><li>Van der Pol, E., B&#246;ing, A. N., Harrison, P., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+functions,+and+clinical+relevance+of+extracellular+vesicles.">Classification functions, and clinical relevance of extracellular vesicles.</a> Pharmacological Reviews. 64 (3), 676-705 (2012).</li><li>Raposo, G., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+Exosomes,+microvesicles,+and+friends.">Extracellular vesicles: Exosomes, microvesicles, and friends.</a> The Journal of Cell Biology. 200 (4), 373-383 (2013).</li><li>Dinkla, S., Brock, R., Joosten, I., Bosman, G. J. C. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway+to+understanding+microparticles:+standardized+isolation+and+identification+of+plasma+membrane-derived+vesicles.">Gateway to understanding microparticles: standardized isolation and identification of plasma membrane-derived vesicles.</a> Nanomedicine (London, England). 8 (10), (2013).</li><li>Witwer, K. W., Buzas, E. I., 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=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> Journal of Extracellular Vesicles. 2, (2013).</li><li>Shah, M. D., Bergeron, A. L., Dong, J. -. F., L&#243;pez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+measurement+of+microparticles:+Pitfalls+and+protocol+modifications.">Flow cytometric measurement of microparticles: Pitfalls and protocol modifications.</a> Platelets. 19 (5), 365-372 (2008).</li><li>Dey-Hazra, E., Hertel, B., 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=Detection+of+circulating+microparticles+by+flow+cytometry:+influence+of+centrifugation,+filtration+of+buffer,+and+freezing.">Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing.</a> Vascular Health and Risk Management. 6, 1125-1133 (2010).</li><li>Lacroix, R., Judicone, C., 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=Standardization+of+pre-analytical+variables+in+plasma+microparticle+determination:+results+of+the+International+Society+on+Thrombosis+and+Haemostasis+SSC+Collaborative+workshop.">Standardization of pre-analytical variables in plasma microparticle determination: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop.</a> Journal of Thrombosis and Haemostasis. 11 (6), 1190-1193 (2013).</li><li>Mullier, F., Bailly, N., Chatelain, C., Chatelain, B., Dogn&#233;, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-analytical+issues+in+the+measurement+of+circulating+microparticles:+current+recommendations+and+pending+questions.">Pre-analytical issues in the measurement of circulating microparticles: current recommendations and pending questions.</a> Journal of Thrombosis and Haemostasis. 11 (4), 693-696 (2013).</li><li>Peramo, P., 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=Physical+Characterization+of+Mouse+Deep+Vein+Thrombosis+Derived+Microparticles+by+Differential+Filtration+with+Nanopore+Filters.">Physical Characterization of Mouse Deep Vein Thrombosis Derived Microparticles by Differential Filtration with Nanopore Filters.</a> Membranes. 2 (1), 1-15 (2012).</li><li>Tilley, R. E., Holscher, T., Belani, R., Nieva, J., Mackman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Factor+Activity+is+Increased+in+a+Combined+Platelet+and+Microparticle+Sample+from+Cancer+Patients.">Tissue Factor Activity is Increased in a Combined Platelet and Microparticle Sample from Cancer Patients.</a> Thrombosis research. 122 (5), 604-609 (2008).</li><li>Rood, I. M., Deegens, J. K. J., 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=Comparison+of+three+methods+for+isolation+of+urinary+microvesicles+to+identify+biomarkers+of+nephrotic+syndrome.">Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.</a> Kidney international. 78 (8), 810-816 (2010).</li><li>Van der Pol, E., Hoekstra, A. G., Sturk, A., Otto, C., van Leeuwen, T. G., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+non-optical+methods+for+detection+and+characterization+of+microparticles+and+exosomes.">Optical and non-optical methods for detection and characterization of microparticles and exosomes.</a> Journal of Thrombosis And Haemostasis: JTH. 8 (12), 2596-2607 (2010).</li><li>Gy&#246;rgy, B., Szab&#243;, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles,+current+state-of-the-art:+emerging+role+of+extracellular+vesicles.">Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.</a> Cellular and Molecular Life Sciences. 68 (16), 2667-2688 (2011).</li><li>Miguet, L., Be&#769;chade, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+Malignant+B-Cell+Derived+Microparticles+Reveals+CD148+as+a+Potentially+Useful+Antigenic+Biomarker+for+Mantle+Cell+Lymphoma+Diagnosis.">Proteomic Analysis of Malignant B-Cell Derived Microparticles Reveals CD148 as a Potentially Useful Antigenic Biomarker for Mantle Cell Lymphoma Diagnosis.</a> Journal of Proteome Research. 8 (7), 3346-3354 (2009).</li><li>Smalley, D. M., Root, K. E., Cho, H., Ross, M. M., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+discovery+of+21+proteins+expressed+in+human+plasma-derived+but+not+platelet-derived+microparticles.">Proteomic discovery of 21 proteins expressed in human plasma-derived but not platelet-derived microparticles.</a> Thrombosis and Haemostasis. 97 (1), 67-80 (2007).</li><li>Lacroix, R., Robert, S., Poncelet, P., Dignat-George, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+Limitations+of+Microparticle+Measurement+by+Flow+Cytometry.">Overcoming Limitations of Microparticle Measurement by Flow Cytometry.</a> Seminars in Thrombosis and Hemostasis. 36 (08), 807-818 (2010).</li><li>Mobarrez, F., Antovic, J., 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=A+multicolor+flow+cytometric+assay+for+measurement+of+platelet-derived+microparticles.">A multicolor flow cytometric assay for measurement of platelet-derived microparticles.</a> Thrombosis Research. 125 (3), e110-e116 (2010).</li><li>Van der Pol, E., van Gemert, M. J. C., Sturk, A., Nieuwland, R., van Leeuwen, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+vs.+swarm+detection+of+microparticles+and+exosomes+by+flow+cytometry.">Single vs. swarm detection of microparticles and exosomes by flow cytometry.</a> Journal of Thrombosis And Haemostasis: JTH. 10 (5), 919-930 (2012).</li><li>Gy&#246;rgy, B., Szab&#243;, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Flow+Cytometric+Assessment+Reveals+Distinct+Microvesicle+(Cell-Derived+Microparticle)+Signatures+in+Joint+Diseases.">Improved Flow Cytometric Assessment Reveals Distinct Microvesicle (Cell-Derived Microparticle) Signatures in Joint Diseases.</a> PLoS ONE. 7 (11), e49726 (2012).</li><li>Gy&#246;rgy, B., M&#243;dos, K., 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=Detection+and+isolation+of+cell-derived+microparticles+are+compromised+by+protein+complexes+resulting+from+shared+biophysical+parameters.">Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters.</a> Blood. 117 (4), e39-e48 (2011).</li><li>Gy&#246;rgy, B., Pasztoi, M., Buzas, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response:+systematic+use+of+Triton+lysis+as+a+control+for+microvesicle+labeling.">Response: systematic use of Triton lysis as a control for microvesicle labeling.</a> Blood. 119 (9), 2175-2176 (2012).</li><li>Trummer, A., De Rop, C., Tiede, A., Ganser, A., Eisert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotype+controls+in+phenotyping+and+quantification+of+microparticles:+a+major+source+of+error+and+how+to+evade+it.">Isotype controls in phenotyping and quantification of microparticles: a major source of error and how to evade it.</a> Thrombosis Research. 122 (5), 691-700 (2008).</li><li>Shet, A. S., Aras, O., 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=Sickle+blood+contains+tissue+factor-positive+microparticles+derived+from+endothelial+cells+and+monocytes.">Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes.</a> Blood. 102 (7), 2678-2683 (2003).</li><li>Connor, D. E., Exner, T., Ma, D. D. F., Joseph, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+circulating+platelet-derived+microparticles+fail+to+bind+annexin+V,+lack+phospholipid-dependent+procoagulant+activity+and+demonstrate+greater+expression+of+glycoprotein+Ib.">The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib.</a> Thrombosis and Haemostasis. 103 (5), 1044-1052 (2010).</li><li>Nolte-&#8217;t Hoen, E. N. M., van der Vlist, E. J., 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=Quantitative+and+qualitative+flow+cytometric+analysis+of+nanosized+cell-derived+membrane+vesicles.">Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles.</a> Nanomedicine: Nanotechnology, Biology, And Medicine. 8 (5), 712-720 (2012).</li><li>Van der Vlist, E. J., Nolte-&#8217;t Hoen, E. N. M., Stoorvogel, W., Arkesteijn, G. J. A., Wauben, M. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeling+of+nano-sized+vesicles+released+by+cells+and+subsequent+quantitative+and+qualitative+analysis+by+high-resolution+flow+cytometry.">Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.</a> Nature Protocols. 7 (7), 1311-1326 (2012).</li><li>Orozco, A. F., Lewis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+circulating+microparticles+in+plasma.">Flow cytometric analysis of circulating microparticles in plasma.</a> Cytometry Part A. 77 A. 77A (6), 502-514 (2010).</li></ol>

الكتاب ليس لديهم تضارب المصالح في الكشف عنها.

وقدمت اثنين من بروتوكولات مختلفة لعزل ومعالجة وتحليل المركبات الكهربائية، وذلك باستخدام إما الكشف الفردي أو النهج القائم على حبة. اختيار أنسب طريقة لاستخدام ليست دائما واضحة ويتطلب فهما للعينة التي يجري اختبارها وكذلك مجموعات سكانية فرعية فردية من الفائدة. وعلاوة ...

خارج الخلية الحويصلات (المركبات الكهربائية) هي، الحويصلات المشتقة من غشاء صغيرة وجدت في سوائل الجسم التي تشارك بشكل كبير في التواصل خلية خلية وتساعد على تنظيم مجموعة متنوعة من العمليات البيولوجية. وقد تم تحليل المركبات الكهربائية باستخدام التدفق الخلوي (FCM) بالغ الصعوبة نظرا لصغر حجمها وقلة السكان منفصلة إيجابي لعلامات من الفائدة. طرق للتحليل EV، في حين تحسنت تحسنا كبيرا على مدى العقد الماضي، لا يزال التقدم في العمل. لسوء الحظ، ليس هناك مقاس واحد يناسب الجميع البروتوكول، ويجب اعتبار العديد من الجوانب عند تحديد أنسب طريقة للاستخدام. المقدمة هنا عدة تقنيات مختلفة لمعالجة المركبات الكهربائية وبروتوكولين لتحليل المركبات الكهربائية باستخدام إما الكشف الفردي أو النهج القائم على حبة. الأساليب المذكورة هنا سوف تساعد في القضاء على المجاميع الأجسام المضادة التي توجد عادة في المستحضرات التجارية، وزيادة نسبة الإشارة إلى الضوضاء، ووضع بوابات في و عقلانيةashion أن يقلل من الكشف عن مضان الخلفية. يستخدم البروتوكول الأول لطريقة الكشف الفردية التي هي خاصة مناسبة تماما لتحليل كميات كبيرة من العينات السريرية، في حين يستخدم بروتوكول الثاني النهج القائم على حبة لالتقاط وكشف المركبات الكهربائية الصغيرة وexosomes. المركبات الكهربائية، المعروف أيضا باسم المجهرية الدقيقة، هي، الحويصلات المشتقة من غشاء صغيرة وجدت في سوائل الجسم التي تشارك في الاتصالات خلية خلية وتساعد على تنظيم مجموعة متنوعة من العمليات البيولوجية 1. من خلال التعبير عن علامات سطح مختلفة و / أو التحويل المباشر للمواد البيولوجية والمركبات الكهربائية قادرة على تغيير وظيفة الخلايا المتلقية للعب إما تفعيل أو قمع الأدوار في التواصل بين الخلايا 2-4. ومن المعروف سريريا، المركبات الكهربائية المستمدة من الصفائح الدموية لديها نشاط مضاد للتخثر قوي 5، في حين تم أظهرت الآخرين للمساهمة في مجموعة واسعة من الشروط، من تعزيز الورم metastaجهاز الأمن والمخابرات 6 إلى حماية ضد مرض 7. ويمكن تصنيف المركبات الكهربائية إلى فئات صغيرة من الحويصلات المشتقة من الخلايا مثل exosomes وmicrovesicles (MVS)، اعتمادا على حجمها وآلية لتوليد 8. تواصل تسمية المجموعات السكانية الفرعية حويصلة المشتقة من خلية ليكون موضوع النقاش الدائر 8،9، ومع ذلك، تم وصفها exosomes عموما صغيرة، 40-100 نانومتر الجزيئات المستمدة من الانصهار endosomal مع غشاء البلازما، في حين MVS تكون أكبر من 100 إلى 1،000 الجسيمات نانومتر التي شكلتها سفك غشاء البلازما 10. هنا، المصطلح العام &quot;السيارات الكهربائية&quot; سوف تستخدم للإشارة إلى جميع أنواع الحويصلات البيولوجية خارج الخلية التي تحررها الخلايا. عزل المركبات الكهربائية من الدم الكامل هو إجراء متعددة الخطوات، وقد أظهرت العديد من المتغيرات معالجة مختلفة للتأثير على المحتوى EV، بما في ذلك درجة حرارة التخزين ومدة 11،12، تخثر / حافظة تستخدم 13 وتستخدم طريقة الطرد المركزي 14. وأدى الحاجة إلى توحيد هذه المتغيرات لتوصيات الجمعية الدولية على تخثر الدم والمعالجة وEV العزلة إجراءات تخثر الدم جنة توحيد القياسي (ISTH SSC) لمناسبة العلمية و15،16، ولكن هناك عدم وجود إجماع بين الباحثين على بروتوكول الأمثل لاستخدام 12. معظم توافق، مع ذلك، أن تخضع لرقابة مشددة متغيرات ما قبل التحليلية حاسمة لبيانات دقيقة وقابلة للتكرار. من أجل تحليل المركبات الكهربائية، واستخدمت الباحثون أساليب مختلفة، بما في ذلك انتقال الإلكترون المجهري 17، المسح الإلكترون المجهري 18،19، مجهر القوة الذرية، وعلى ضوء ديناميكية نثر 20،21 والغربية النشاف 22،23. في حين FCM هو الأسلوب المفضل لكثير من الباحثين 9،24 - 26 نظرا لقدراتها العالية الإنتاجية، وقد تم تحليل المركبات الكهربائية باستخدام FCMمن الصعب المعروف نظرا لحجمها وقلة السكان إيجابي منفصلة 27 - 32. مقارنة تحليل الخلايا، حجم صغير من النتائج المركبات الكهربائية في 1) أقل مضان المنبعثة نتيجة لعدد أقل من المستضدات في الجسيمات و2) الجدوى محدود من الغسيل بعد وصمة عار، وهو أمر ضروري للحد من مضان الخلفية. التحديات المشتركة بين الباحثين وتشمل إشارات الناشئة عن المجاميع المناعي 27،28 والتجميع الذاتي للأجسام 29. وعلاوة على ذلك، أوقات المعالجة طويلة ومطولة إجراءات الغسيل / العزلة التي يستخدمها العديد من البروتوكولات الحالية 33،34 تتطلب التزامات الوقت عدة أيام لتحليل عدد قليل من العينات، مما يجعلها أقل من مثالية لتطبيقات إنتاجية عالية. بعض الباحثين التخلي عن خطوة غسل تماما، مما يجعل المستخدمة تقليديا FCM ضوابط السلبية مثل مضان ناقص واحد (FMO) وisotypes الأجسام المضادة غير مجدية لتقييم بدقة البكالوريامضان kground 30. بروتوكولات عنوان لدينا ثلاث مشاكل المشتركة التي يمكن أن تعوق تحليل FCM السليم من المركبات الكهربائية: إشارات الناشئة عن المجاميع الأجسام المضادة وغيرها من غير الحويصلات، صعوبة في إزالة الأجسام المضادة غير منضم، وعدم وجود السكان إيجابي ملحوظ. سوف التقنيات الموضحة هنا مساعدة في القضاء على المجاميع الأجسام المضادة التي توجد عادة في المستحضرات التجارية، وزيادة نسبة الإشارة إلى الضوضاء، ووضع البوابات بطريقة عقلانية أن يقلل من الكشف عن مضان الخلفية. يتم عرض اثنين من طرق الكشف مختلفة هنا: يستخدم البروتوكول الأول لطريقة الكشف الفردية التي هي خاصة مناسبة تماما لتحليل كميات كبيرة من العينات السريرية، في حين يستخدم بروتوكول الثاني النهج القائم على حبات لالتقاط وكشف المركبات الكهربائية الصغيرة وexosomes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >LSR II benchtop flow cytometer</td>
			<td >BD Biosciences</td>
			<td ></td>
			<td >3-laser (20 mW Coherent Sapphire 488 nm blue, 25 mW Coherent Vioflame 405 nm violet, and 17 mW JDS Uniphase HeNe 633nm red)</td>
		</tr>
		<tr >
			<td >FACS Diva software&#160;</td>
			<td >BD Biosciences</td>
			<td ></td>
			<td >PC version 6.0</td>
		</tr>
		<tr >
			<td >FlowJo software&#160;</td>
			<td >Treestar US</td>
			<td ></td>
			<td >Mac version 9.6.1 or PC version 7.6.5</td>
		</tr>
		<tr >
			<td >Sphero Rainbow fluorescent particles</td>
			<td >BD Biosciences</td>
			<td >556298</td>
			<td >used to adjust all channel voltages to maintain fluorescence intensity consistency&#160;</td>
		</tr>
		<tr >
			<td >Ultra Rainbow fluorescent particles&#160;</td>
			<td >Spherotech</td>
			<td >URFP-10-5</td>
			<td >used in addition to Megamix-Plus SSC beads to ensure EV gating consistency from batch to batch</td>
		</tr>
		<tr >
			<td >Megmix-Plus SSC beads</td>
			<td >Biocytex</td>
			<td >7803</td>
			<td >used to adjust FSC and SSC&#160; voltages to maintain&#160; consistency&#160; between runs. Can also used to monitor flow rate and ajust flow rate dial in order to ensure that same flow rate is used in all runs</td>
		</tr>
		<tr >
			<td >AbC Anti-Mouse Bead Kit</td>
			<td >Life Technologies</td>
			<td >A-10344</td>
			<td >used for compensation controls &#38; negative AbC beads used for beads-based method</td>
		</tr>
		<tr >
			<td >Nonidet P-40 Alternative (NP-40) (CAS 9016-45-9)</td>
			<td >Santa Cruz&#160;</td>
			<td >sc-281108</td>
			<td >used in the individual detection method only to lyse samples after initial reading for use as negative controls. Stock may be diluted to 1:10 in PBS and stored in fridge for up to 1 month.</td>
		</tr>
		<tr >
			<td >BD TruCOUNT Tubes</td>
			<td >BD Biosciences</td>
			<td >340334</td>
			<td >used whenver absolute EV concentrations are needed</td>
		</tr>
		<tr >
			<td >Ultrafree-MC, GV 0.22 &#181;m Centrifugal Filter Units</td>
			<td >Millipore&#160;</td>
			<td >UFC30GVNB</td>
			<td >used to post-stain wash Evs and/or fractionate EVs based on size</td>
		</tr>
		<tr >
			<td >Vacutainer glass whole blood tubes ACD-A</td>
			<td >BD Biosciences</td>
			<td >364606</td>
			<td ></td>
		</tr>
		<tr >
			<td >Facs tubes 12x75 polystrene</td>
			<td >BD Biosciences</td>
			<td >352058</td>
			<td ></td>
		</tr>
		<tr >
			<td >50mL Reservoirs individually wrapped&#160;</td>
			<td >Phenix</td>
			<td >RR-50-1s</td>
			<td ></td>
		</tr>
		<tr >
			<td >Green-Pak pipet tips - 10&#181;L</td>
			<td >Rainin</td>
			<td >GP-L10S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Green-Pak pipet tips -200&#181;L&#160;</td>
			<td >Rainin</td>
			<td >GP-L250S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Green -Pak pipet tips - 1000&#181;L&#160;</td>
			<td >Rainin</td>
			<td >GP-L1000S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Stable Stack L300 tips presterilized</td>
			<td >Rainin</td>
			<td >SS-L300S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Pipet-Lite&#160;XLS 8 Channel LTS&#160;Adjustable&#160;Spacer&#160;</td>
			<td >Rainin</td>
			<td >LA8-300XLS</td>
			<td ></td>
		</tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td >E&#38;K Scientific</td>
			<td >EK-20180</td>
			<td ></td>
		</tr>
		<tr >
			<td >RPMI 1640 Media (without Hepes)</td>
			<td >UCSF Cell Culture Facility</td>
			<td >CCFAE001</td>
			<td >media used for bead-based detection method</td>
		</tr>
		<tr >
			<td >Dulbeccos PBS D-PBS, CaMg-free, 0.2&#181;m filtered</td>
			<td >UCSF Cell Culture Facility</td>
			<td >CCFAL003</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ultracentrifuge Tube, Thinwall, Ultra-Clear</td>
			<td >BECKMAN COULTER INC</td>
			<td >344058</td>
			<td ></td>
		</tr>
		<tr >
			<td >PANEL I</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >CD3 PerCP-Cy5.5</td>
			<td >Biolegend</td>
			<td >344808</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD14 APC-Cy7</td>
			<td >Biolegend</td>
			<td >301820</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD16 V450</td>
			<td >BD Biosciences</td>
			<td >560474</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD28 FITC</td>
			<td >biolegend</td>
			<td >302906</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD152 APC</td>
			<td >BD Biosciences</td>
			<td >555855</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD19 A700</td>
			<td >Biolegend</td>
			<td >302226</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >PANEL II</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >CD41a PerCP-Cy5.5</td>
			<td >BD Biosciences</td>
			<td >340930</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD62L APC</td>
			<td >Biolegend</td>
			<td >304810</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD108 PE&#160;</td>
			<td >BD Biosciences</td>
			<td >552830</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD235a FITC</td>
			<td >biolegend</td>
			<td >349104</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >PANEL III</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >CD11b PE-Cy7</td>
			<td >Biolegend</td>
			<td >301322</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD62p APC</td>
			<td >Biolegend</td>
			<td >304910</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD66b PE&#160;</td>
			<td >Biolegend</td>
			<td >305106</td>
			<td >2 &#181;l</td>
		</tr>
		<tr >
			<td >CD15 FITC</td>
			<td >exalpha</td>
			<td >X1496M</td>
			<td >5 &#181;l</td>
		</tr>
		<tr >
			<td >CD9 PE</td>
			<td >Biolegend</td>
			<td >555372</td>
			<td ></td>
		</tr>
		<tr >
			<td >CD63 APC</td>
			<td >Biolegend</td>
			<td >353008</td>
			<td ></td>
		</tr>
		<tr >
			<td >APC-Cy7 Ms IgG2a, &#954;</td>
			<td >Biolegend</td>
			<td >400230</td>
			<td ></td>
		</tr>
	</tbody>
</table>

techniques for the analysis of extracellular vesicles using flow cytometry

خارج الخلية الحويصلات (المركبات الكهربائية) هي، الحويصلات المشتقة من غشاء صغيرة وجدت في سوائل الجسم التي تشارك بشكل كبير في التواصل خلية خلية وتساعد على تنظيم مجموعة متنوعة من العمليات البيولوجية. وقد تم تحليل المركبات الكهربائية باستخدام التدفق الخلوي (FCM) بالغ الصعوبة نظرا لصغر حجمها وقلة السكان منفصلة إيجابي لعلامات من الفائدة. طرق للتحليل EV، في حين تحسنت تحسنا كبيرا على مدى العقد الماضي، لا يزال التقدم في العمل. لسوء الحظ، ليس هناك مقاس واحد يناسب الجميع البروتوكول، ويجب اعتبار العديد من الجوانب عند تحديد أنسب طريقة للاستخدام. المقدمة هنا عدة تقنيات مختلفة لمعالجة المركبات الكهربائية وبروتوكولين لتحليل المركبات الكهربائية باستخدام إما الكشف الفردي أو النهج القائم على حبة. الأساليب المذكورة هنا سوف تساعد في القضاء على المجاميع الأجسام المضادة التي توجد عادة في المستحضرات التجارية، وزيادة نسبة الإشارة إلى الضوضاء، ووضع بوابات في و عقلانيةashion أن يقلل من الكشف عن مضان الخلفية. يستخدم البروتوكول الأول لطريقة الكشف الفردية التي هي خاصة مناسبة تماما لتحليل كميات كبيرة من العينات السريرية، في حين يستخدم بروتوكول الثاني النهج القائم على حبة لالتقاط وكشف المركبات الكهربائية الصغيرة وexosomes.

<html> ويبين الشكل 1 الخطة الشاملة لعزل وكشف عن المركبات الكهربائية باستخدام إما طريقة القائم على حبة أو طريقة الكشف الفردية المعالجة. كشف الفردية من المركبات الكهربائية باستخدام FCM يعمل بشكل جيد لتحليل المركبات الكهربائية الكبيرة إلا أن معظم cytometers ليست قادرة ...

Watch this Scientific Journal Video about Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry at JoVE.com

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

وجود العديد من الطرق المختلفة لقياس الحويصلات خارج الخلية (المركبات الكهربائية) باستخدام التدفق الخلوي (FCM). وينبغي النظر في العديد من الجوانب عند تحديد أنسب طريقة للاستخدام. يتم عرض بروتوكولين لقياس المركبات الكهربائية، وذلك باستخدام إما الكشف الفردي أو النهج القائم على حبة.

تقنيات لتحليل خارج الخلية الحويصلات عن طريق التدفق الخلوي

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

techniques-for-analysis-extracellular-vesicles-using-flow

Research

JoVE Journal

Biology

22.9K Views.  Blood Systems Research Institute.  وجود العديد من الطرق المختلفة لقياس الحويصلات خارج الخلية (المركبات الكهربائية) باستخدام التدفق الخلوي (FCM). وينبغي النظر في العديد من الجوانب عند تحديد أنسب طريقة للاستخدام. يتم عرض بروتوكولين لقياس المركبات الكهربائية، وذلك باستخدام إما...

Video: Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

تدفق تنقية الخلوي للخلايا الفأر الانتصافي

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

الأمثل أساليب النمذجة وتلوين لرصد انتشار استخدام الأصباغ انقسام الخلية خلية تتبع

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

تتبع الزمني للتقدم دورة الخلية باستخدام التدفق الخلوي دون الحاجة إلى مزامنة

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

إعداد نموذج لتحليل قداس الخلوي

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED الحرارية تدفق - الجمع بين علم البصريات الوراثي مع التدفق الخلوي

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

تحليل معلقات خلية معزولة من الأنسجة الصلبة من قبل الطيفي التدفق الخلوي

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

تحليل سيتوميتريك من حويصلات خارج الخلية من خلية مكيفة وسائل الإعلام تدفق

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

تنقية وتحليل Caenorhabditis elegans خارج الخلية الحويصلات

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

تحليل تتبع الجسيمات النانوية لتحديد كمي وحجم الحويصلات خارج الخلية

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

توصيف البروتين عبر الغشاء الحويصلة خارج الخلية بواسطة قياس التدفق الخلوي النانوي

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...