Name: characterization of human monocyte-derived dendritic cells by imaging flow cytometry: a comparison between two monocyte isolation protocols
Uploaded: 2016-10-18T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols at JoVE.com

This research is supported by the National Institute on Alcohol Abuse and Alcoholism, award K99/R00 AA021264. Additional lab support as part of startup package has been received from the Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, and FIU- Office of Research and Economic Development. Gianna Casteleiro was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

В целом исследования крови человека были рассмотрены и одобрены Советом по институциональному (IRB) ПФР, IRB протокол утверждения # IRB-13-0440. Человеческие leukopaks были приобретены у банковского сообщества крови в Майами, штат Флорида. 1. Выделение МКПК с помощью стандартной плотн...

<ol>
	<li>Cella, M., Sallusto, F., Lanzavecchia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+maturation+and+antigen+presenting+function+of+dendritic+cells.">Origin maturation and antigen presenting function of dendritic cells.</a> Curr Opin Imunol. 9, 10-16 (1997).</li><li>Banchereau, 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=Immunobiology+of+Dendritic+Cells.">Immunobiology of Dendritic Cells.</a> Ann Rev Immunol. 18, 767-811 (2000).</li><li>Banchereau, J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+the+control+of+immunity.">Dendritic cells and the control of immunity.</a> Nature. 392, 245-252 (1998).</li><li>Kaouther, M., Ridha, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Cell-Based+Graft+Tolerance.">Dendritic Cell-Based Graft Tolerance.</a> ISRN Pharmacol. , (2011).</li><li>Steinman, R., Gutchinov, B., Witmer, M., Nussenzweig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+are+the+principal+stimulators+of+the+primary+mixed+leukocyte+reaction+in+mice.">Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice.</a> J Exp Med. 157, 613-627 (1983).</li><li>Nair, M. N., 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=RNAi-directed+inhibition+of+DC-SIGN+by+dendritic+cells:+Prospects+for+HIV-1+therapy.">RNAi-directed inhibition of DC-SIGN by dendritic cells: Prospects for HIV-1 therapy.</a> AAPS J. 7, E572-E578 (2005).</li><li>Agudelo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+10.">Chapter 10.</a> Dendritic Cells: Types, Life Cycles and Biological Functions. , 167-177 (2010).</li><li>Kajihara, M., Takakura, K., Ohkusa, T., Koido, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+dendritic+cell-tumor+fusion+cells+on+cancer+vaccines+-+past+progress+and+future+strategies.">The impact of dendritic cell-tumor fusion cells on cancer vaccines - past progress and future strategies.</a> Immunotherapy. , (2015).</li><li>Suwandi, J., Toes, R., Nikolic, T., Roep, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+tissue+specific+tolerance+in+autoimmune+disease+with+tolerogenic+dendritic+cells.">Inducing tissue specific tolerance in autoimmune disease with tolerogenic dendritic cells.</a> Clin Exp Rheumatol. 33, 0097-0103 (2015).</li><li>Gorelik, M., Frischmeyer-Guerrerio, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+dendritic+cell+responses+to+immunotherapy.">Innate and adaptive dendritic cell responses to immunotherapy.</a> Curr Opin Allergy Immunol. 15, 575-580 (2015).</li><li>Zwolak, 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=Peripheral+blood+dendritic+cells+in+alcoholic+and+autoimmune+liver+disorders.">Peripheral blood dendritic cells in alcoholic and autoimmune liver disorders.</a> Hum Exp Toxicol. 31, 438-446 (2012).</li><li>Agudelo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+functional+role+of+cannabinoid+genes+in+alcohol+users.">Differential expression and functional role of cannabinoid genes in alcohol users.</a> Drug Alcohol Depend. 133, 789-793 (2013).</li><li>Nair, M. P., Figueroa, G., Casteleiro, G., Mu&#241;oz, K., Agudelo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+Versus+Cannabinoids:+A+Review+of+Their+Opposite+Neuro-Immunomodulatory+Effects+and+Future+Therapeutic+Potentials.">Alcohol Versus Cannabinoids: A Review of Their Opposite Neuro-Immunomodulatory Effects and Future Therapeutic Potentials.</a> J Alcohol Drug Depend. 3, 184 (2015).</li><li>Boukli, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+ER+Stress,+the+Unfolded+Protein+Response,+and+Pro-+and+Anti-Apoptotic+Protein+Fingerprints+in+Human+Monocyte-Derived+Dendritic+Cells+Treated+With+Alcohol.">Implications of ER Stress, the Unfolded Protein Response, and Pro- and Anti-Apoptotic Protein Fingerprints in Human Monocyte-Derived Dendritic Cells Treated With Alcohol.</a> Alcohol Clin Exp Res. 34, 2081-2088 (2010).</li><li>Nair, M. N., Mahajan, S., Sykes, D., Bapardekar, M., Reynolds, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+Modulates+DC-SIGN+Expression+by+Mature+Dendritic+Cells.">Methamphetamine Modulates DC-SIGN Expression by Mature Dendritic Cells.</a> J Neuroimmune Pharmacol. 1, 296-304 (2006).</li><li>Napuri, 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=Cocaine+Enhances+HIV-1+Infectivity+in+Monocyte+Derived+Dendritic+Cells+by+Suppressing+microRNA-155.">Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155.</a> PLoS ONE. 8, e83682 (2013).</li><li>Nair, M. P. N., Saiyed, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+methamphetamine+on+expression+of+HIV+coreceptors+and+CC-chemokines+by+dendritic+cells.">Effect of methamphetamine on expression of HIV coreceptors and CC-chemokines by dendritic cells.</a> Life Sciences. 88, 987-994 (2011).</li><li>Nair, M. P. N., 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=Cocaine+Modulates+Dendritic+Cell-Specific+C+Type+Intercellular+Adhesion+Molecule-3-Grabbing+Nonintegrin+Expression+by+Dendritic+Cells+in+HIV-1+Patients.">Cocaine Modulates Dendritic Cell-Specific C Type Intercellular Adhesion Molecule-3-Grabbing Nonintegrin Expression by Dendritic Cells in HIV-1 Patients.</a> J Immunol. 174, 6617-6626 (2005).</li><li>Reynolds, J. L., Mahajan, S. D., Sykes, D. E., Schwartz, S. A., Nair, M. P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analyses+of+methamphetamine+(METH)-induced+differential+protein+expression+by+immature+dendritic+cells+(IDC).">Proteomic analyses of methamphetamine (METH)-induced differential protein expression by immature dendritic cells (IDC).</a> Biochem Biophys Acta. 1774, 433-442 (2007).</li><li>Van Voorhis, W., Hair, L., Steinman, R., Kaplan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dendritic+cells.+Enrichment+and+characterization+from+peripheral+blood.">Human dendritic cells. Enrichment and characterization from peripheral blood.</a> J Exp Med. 155, 1172-1187 (1982).</li><li>Davis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mac-1+and+p150,95+beta+2+integrins+bind+denatured+proteins+to+mediate+leukocyte+cell-substrate+adhesion.">The Mac-1 and p150,95 beta 2 integrins bind denatured proteins to mediate leukocyte cell-substrate adhesion.</a> Exp Cell Res. 200, 242-252 (1992).</li><li>Delirezh, N., Shojaeefar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+comparison+between+flask+adherent+and+magnetic+activated+cell+sorted+monocytes+derived+dendritic+cells.">Phenotypic and functional comparison between flask adherent and magnetic activated cell sorted monocytes derived dendritic cells.</a> Iran J Immunol. 9, 98-108 (2012).</li><li>Lehner, M., Holter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin-Free+Purification+of+Monocytes+for+Dendritic+Cell+Generation+via+Discontinuous+Density+Gradient+Centrifugation+Based+on+Diluted+Ficoll-Paque+Plus&lt;sup&gt;&#174;&lt;/sup&gt;.">Endotoxin-Free Purification of Monocytes for Dendritic Cell Generation via Discontinuous Density Gradient Centrifugation Based on Diluted Ficoll-Paque Plus&lt;sup&gt;&#174;&lt;/sup&gt;.</a> Int Arch Allergy Immunol. 128, 73-76 (2002).</li><li>Van Brussel, 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=Fluorescent+activated+cell+sorting:+An+effective+approach+to+study+dendritic+cell+subsets+in+human+atherosclerotic+plaques.">Fluorescent activated cell sorting: An effective approach to study dendritic cell subsets in human atherosclerotic plaques.</a> J. Immunol Methods. 417, 76-85 (2015).</li><li>Mucci, 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=The+methodological+approach+for+the+generation+of+humandendritic+cells+from+monocytes+affects+the+maturation+state+of+the+resultant+dendritic+cells.">The methodological approach for the generation of humandendritic cells from monocytes affects the maturation state of the resultant dendritic cells.</a> Biologicals. 37, 288-296 (2009).</li><li>Yuan, N., 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> The American Association of Immunologists (AAI). , (2007).</li><li>Romani, N., 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=Proliferating+dendritic+cell+progenitors+in+human+blood.">Proliferating dendritic cell progenitors in human blood.</a> J Exp Med. 180, 83-93 (1994).</li><li>Bennett, S., Breit, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variables+in+the+isolation+and+culture+of+human+monocytes+that+are+of+particular+relevance+to+studies+of+HIV.">Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV.</a> J Leukoc Biol. 56, 236-240 (1994).</li><li>Gr&#252;tzkau, A., Radbruch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+but+mighty:+How+the+MACS&#174;-technology+based+on+nanosized+superparamagnetic+particles+has+helped+to+analyze+the+immune+system+within+the+last+20+years.">Small but mighty: How the MACS&#174;-technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years.</a> Cytometry Part A. 77A, 643-647 (2010).</li><li>El-Sahrigy, S. A., Mohamed, N. A., Talkhan, H. A., Rahman, A. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+magnetic+activated+cell+sorted+monocytes+and+monocyte+adherence+techniques+for+in+vitro+generation+of+immature+dendritic+cells:+an+Egyptian+trial.">Comparison between magnetic activated cell sorted monocytes and monocyte adherence techniques for in vitro generation of immature dendritic cells: an Egyptian trial.</a> Cent Eur J Immunol. 40, 18-24 (2015).</li><li>Curry, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differential Blood Count. , (2015).</li><li>Sallusto, F., Lanzavecchia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+presentation+of+soluble+antigen+by+cultured+human+dendritic+cells+is+maintained+by+granulocyte/macrophage+colony-stimulating+factor+plus+interleukin+4+and+downregulated+by+tumor+necrosis+factor+alpha.">Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.</a> J Exp Med. 179, 1109-1118 (1994).</li><li>Cavanagh, L. L., Saal, R. J., Grimmett, K. L., Thomas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+in+Monocyte-Derived+Dendritic+Cell+Cultures+Is+Caused+by+Progenitor+Cells+Capable+of+Myeloid+Differentiation.">Proliferation in Monocyte-Derived Dendritic Cell Cultures Is Caused by Progenitor Cells Capable of Myeloid Differentiation.</a> Blood. 92, 1598-1607 (1998).</li><li>Ardeshna, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-derived+dendritic+cells+do+not+proliferate+and+are+not+susceptible+to+retroviral+transduction.">Monocyte-derived dendritic cells do not proliferate and are not susceptible to retroviral transduction.</a> Br J Haematol. 108, 817-824 (2000).</li><li>Chapuis, F., 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=Differentiation+of+human+dendritic+cells+from+monocytes+in+vitro.">Differentiation of human dendritic cells from monocytes in vitro.</a> Eur J Immunol. 27, 431-441 (1997).</li><li>Zhou, L. J., Tedder, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD14++blood+monocytes+can+differentiate+into+functionally+mature+CD83++dendritic+cells.">CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.</a> Proc Natl Acad Sci. 93, 2588-2592 (1996).</li><li>Caux, 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=B70/B7-2+is+identical+to+CD86+and+is+the+major+functional+ligand+for+CD28+expressed+on+human+dendritic+cells.">B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells.</a> J Exp Med. 180, 1841-1847 (1994).</li><li>Caux, 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=Activation+of+human+dendritic+cells+through+CD40+cross-linking.">Activation of human dendritic cells through CD40 cross-linking.</a> J Exp Med. 180, 1263-1272 (1994).</li><li>Fujii, S. i., Liu, K., Smith, C., Bonito, A. J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Linkage+of+Innate+to+Adaptive+Immunity+via+Maturing+Dendritic+Cells+In+Vivo+Requires+CD40+Ligation+in+Addition+to+Antigen+Presentation+and+CD80/86+Costimulation.">The Linkage of Innate to Adaptive Immunity via Maturing Dendritic Cells In Vivo Requires CD40 Ligation in Addition to Antigen Presentation and CD80/86 Costimulation.</a> J Exp Med. 199, 1607-1618 (2004).</li><li>Mohammadi, A., Mehrzad, J., Mahmoudi, M., Schneider, M., Haghparast, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+culture+and+maturation+on+human+monocyte-derived+dendritic+cell+surface+markers,+necrosis+and+antigen+binding.">Effect of culture and maturation on human monocyte-derived dendritic cell surface markers, necrosis and antigen binding.</a> Biotech Histochem. 90, 445-452 (2015).</li></ol>

На основании известных трудностей выделения и генерации MDDCs из человеческой крови, настоящее исследование с целью обеспечить всестороннее сравнение двух хорошо известных методов для генерации MDDCs. Первый способ по сравнению является хорошо установленный традиционный способ генерац?...

Дендритные клетки (ДК) являются важными медиаторами врожденной и адаптивной системы иммунитета. Они действуют, чтобы вызвать первичные иммунные реакции и способствуют развитию иммунологической памяти. Эти клетки в первую очередь ответственны за захват антигена, миграции и стимуляции Т - клеток и, следовательно , называют профессиональных антиген - представляющих клеток (АРС) 1 .Manipulation ДК могут быть использованы в самых различных областях исследований и в клинических условиях для лечения различных воспалительных заболеваний , таких как ВИЧ , 6,7, рак 8, аутоиммунные заболевания 9, а также аллергические реакции 10. Контроллеры домена также используются для исследования злоупотреблением вещества для того , чтобы решить неизвестные механизмы и пути , такие как те , которые связаны с алкогольной зависимостью 11-14 наркотической зависимости 13,15, и сочетание инфекции и наркомании ВИЧ 16-19. Эти текущие исследования и будущие научные исследования Iп области иммунологии делают в пробирке поколения РС чрезвычайно важны для исследования. Тем не менее, существуют некоторые трудности , связанные с выделением контроллеров домена из крови человека , поскольку они составляют лишь 0,1 - 1% от общего количества мононуклеарных клеток крови 20. На сегодняшний день некоторые из хорошо известных методов для генерации РС экстракорпорального состоит из пластика или стекла адгезии моноцитов 21,22, центрифугирования в градиенте плотности 23, удельная разделения на основе маркеров , таких как магнитная активированной сортировки клеток 22, флуоресцентная сортировка клеток 24, положительный выбор CD14 + моноцитов с использованием декстрана покрытием магнитных наночастиц 25 и быстрое выделение высокоочищенных моноцитов с использованием полностью автоматизированной негативной селекции 26 клеток. Тем не менее, лучшим методом выбора остается спорным. Таким образом, для улучшения методов генерации постоянного тока, несколько методов были разработаны, в которыхЧистота этих клеток может быть значительно увеличена путем дифференцировки из очищенных CD34 + клеток - предшественников и моноцитов , выделенных из мононуклеарных клеток периферической крови (МКПК) 27. Как упоминалось до, широко используемый и популярный метод для генерации моноцитов дендритных клеток (MDDCs) заключается в изучении способности моноцитов придерживаться стекла или пластика (метод спаек) 21,22,27. Метод прилипание является быстрым и простым способом, который не требует использования сложного оборудования. Тем не менее, некоторые недостатки этого процесса включают загрязнение лимфоцит, низкую гибкость и моноцитов переходную манипуляцию 28. Альтернативный метод метода прилипание магнитная изоляция моноцитов от общего МНПК, в частности , с использованием человеческого набора обогащения моноцитов, который предназначен для выделения моноцитов МНПК путем негативного отбора 26. Во время этой процедуры нежелательные клетки предназначены для удаления с тетрамернойантитело и магнитные частицы, покрытой декстраном. Преимущество этого способа выделения является то, что нежелательные меченые клетки отделяются с помощью магнита в то время как клетки-мишени могут быть свободно слили в новую пробирку без необходимости колонн. На сегодняшний день, при наличии специфических моноклональных антител, которые могут маркируют уникальные клеточные популяции, метод магнитной сепарации становится не просто дополнительный метод, а необходимость для выделения редких клеток в области иммунологии. Например, методы , такие как магнитно сортировки клеток с коммерчески доступными парамагнитных MACS-наночастиц способствовали разработке новых подходов для проведения исследований и клинических применений 22,29. Кроме того, последние научные исследования , сравнивающие поколение постоянного тока от прилипания моноцитов и технологии MACS методы продемонстрировали более высокую чистоту и жизнеспособность DC с использованием MACS разделенных моноциты 22,30. Настоящее исследование представляетсравнение между двумя методами генерации человеческих моноцитов из ГЦ, выделенных из МНПК: 1) моноцитов изоляции путем присоединения и 2) моноцитов изоляции путем негативной селекции с использованием коммерческого набора обогащения моноцитов человека. Это исследование свидетельствует о том, чтобы показать, что отрицательная магнитная процедура разделения выбора для выделения моноцитов генерирует наибольший выход моноцитов без каких-либо существенных различий в жизнеспособности моноцитов по сравнению с моноцитами, выделенных методом прилипания. В свою очередь, через семь дней, моноциты, выделенные магнитной сепарации дифференцировались в MDDCs со значительно более высоким пролиферативным потенциалом и более высоким количеством клеток, экспрессирующих дважды положительных (CD11c + / CD14 +) фенотипа, не затрагивая жизнеспособность MDDC. В целом, данное исследование отличается от предыдущих исследований, упоминаемых выше, так как он демонстрирует способность обоих методов, чтобы одновременно генерировать моноциты, которые способны пролиферировать и дифференцироваться в CD11c +MDDCs (&gt; 70%) после семи дней в культуре без ущерба для их жизнеспособности. Кроме того, в настоящее время подход предусматривает впервые характеристики различных популяций CD11c / CD14 MDDCs методом проточной цитометрии изображений. Таким образом, так как контроллеры домена играют роль фокусного отношении проведения исследований в области иммунологии, различные параметры должны быть приняты во внимание при рассмотрении вопроса о том , как они получены , и какие методы используются для выделения и культуры их в пробирке. Таким образом, данное исследование направлено на дать представление о двух различных методов выделения моноцитов и как эти методы дифференцированно влияют на жизнеспособность моноцитов и выход в конечном счете, затрагивающего дендритных жизнеспособность клеток, пролиферации и фенотип. Эти результаты будут в значительной степени способствовать области иммунологии и предоставит подробный протокол постоянного выделения, очистки и характеристики.

<table border="0" cellpadding="0" cellspacing="0" width="1435">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:535px;">Ficoll-Paque</td>
			<td style="width:228px;">GE Healthcare</td>
			<td style="width:231px;">17-5442-03</td>
			<td style="width:443px;">Must be used at room temperature</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Phosphate-buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">ACK Lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">RPMI 1640 medium</td>
			<td>Life Technologies</td>
			<td>22400-089</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Antibiotic-Antimycotic (100X)</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">RoboSep buffer</td>
			<td>StemCell</td>
			<td>20104</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">EasySep Human monocyte enrichment kit</td>
			<td>StemCell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0101</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FITC-Dextran</td>
			<td>Sigma Aldrich</td>
			<td>FD4-100MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Trypan blue stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Synergy 2 multi-mode reader</td>
			<td>Biotek</td>
			<td>7131000</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">XTT Sodium salt bioreagent (XTT)</td>
			<td>Sigma Aldrich</td>
			<td>X4626-100MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Dimethyl sulfoxide bioreagent (DMS)</td>
			<td>Sigma Aldrich</td>
			<td>D8418-500ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Thiazolyl blue tetrazolium bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>m5655-500MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td>Bio-Rad</td>
			<td>161-0302</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Phenazine Methosulfate (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>P9626-1G</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Inactivated (HI) Human Serum</td>
			<td>Chemicon</td>
			<td>S1-100ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Accuri C6 Flow Cytometer</td>
			<td>BD Accuri</td>
			<td>653119</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FlowSight Amnis Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>100300</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of human monocyte-derived dendritic cells by imaging flow cytometry: a comparison between two monocyte isolation protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

<html> Моноцитов Выход магнитной сепарацией выше по сравнению с моноцитов Выход по методу ПРИСОЕДИНЕНИЯ Данные , представленные на рисунке 1 отображения РВМС и подсчета клеток моноцитов с помощью трипанового ?...

Watch this Scientific Journal Video about Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols at JoVE.com

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Характеристика человека моноцитов дендритные клетки путем обработки изображений проточной цитометрии: Сравнение между двумя моноцитов Isolation протоколов

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

The overall goal of this study is to compare two different methods of human monocyte isolation for dendritic cell generation in vitro and to characterize the CT11c, CT14 cell surface expression of the resulting monocyte derived populations by imaging flow cytometry. These monocytes isolation protocols can contribute to the development of reliable methods for the purification of human dendritic cells for research and clinical applications. The main advantage of these protocols is that they facilitate the isolation of human monocytes and their differentiation into viable and functional monocyte derived cells.In addition, this is the first study to characterize the specific population of monocyte derived dendritic cells by single cell imaging flow cytometry. This method can also be applied as an alternative for the isolation of other rare cells at an adequate yield for downstream research applications. Visual demonstration of these methods is critical as it can be challenging to handle the blood samples safely without the appropriate instruction and experience.Begin by diluting the blood sample to a one to one ratio with PBS in a T75 flask. Then, add 15 milliliters of density gradient solution into a 50 milliliter centrifuge tube and carefully layer 25 to 30 milliliters of the diluted blood over the gradient. To achieve a proper separation of the peripheral blood mononuclear cells, load the blood onto the density gradient solution quickly, but carefully and without mixing the layers.Separate the cells by centrifugation, followed by the transfer of the white blood cell interface layer into a new 50 milliliter conical tube. Wash the cells two times in PBS, re-suspending the final pellet in 10 milliliters of ammonium chloride potassium lysing buffer for 15 minutes at 4 degrees celsius. Then wash the cells two more times in PBS.To isolate monocytes by the adherence method, culture five times 10 to the seven cells of the resulting PBMC pellet per 10 milliliters of complete medium in a new T75 flask for two hours at 37 degrees celsius and five percent carbon dioxide in a humidified incubator. At the end of the incubation, discard the non-adherent floating cells and gently wash the adherent cells two times with PBS. Then, feed the adherent cells with complete cell culture medium supplemented with human GMCSF and IL4 and return the culture to the incubator for five to seven days.To isolate the monocytes by magnetic separation, after collecting the PBMC by density gradient separation transfer the white blood cell layer to a five milliliter polystyrene tube and re-suspend the cells in PBS at a five times 10 to seven cells per milliliter concentration. Next, add 50 microliters of a human monocyte enrichment cocktail per milliliter to the cells with mixing and incubate the cells with the cocktail at four degrees celsius for 10 minutes. Then add 50 microliters of magnetic particles per milliliter of cells with careful mixing and incubate the cells at 4 degrees celsius for five minutes.At the end of the second incubation, add PBS to the cells to bring the total volume up to two point five milliliters and mix by pipetting two to three times. Then place the polystyrene tube in an appropriate magnetic device at room temperature. After two and a half minutes, with the tube still in the magnet, decant the purified monocyte fraction into a new 15 milliliter conical tube.Then culture the purified monocytes and complete cell culture medium supplemented with GMCSF and IL4 for five to seven days as just demonstrated. After seven days of differentiation, dispense one times 10 to the six cell aliquats of the monocyte derived dendritic cells into the appropriate number of one point five milliliter tubes, and add 50 microliters of heat inactivated human serum to each sample to block any non-specific finding. After 10 minutes, centrifuge the cells and re-suspend the pellets in the appropriate fluorescence conjugated primary antibodies for 20 minutes at four degrees celsius protected from light.Then wash the cells two times in one milliliter of PBS, resuspending the pellets in 100 microliters of PBS per one times ten to the six cells. Just before the analysis, add one microliter of DAPI to each tube. Then, read the samples on a single cell imaging flow cytometer according to the manufacturer's instructions.On average, monocytes isolated by the adherence method account for approximately six point two percent of the total peripheral blood monocyte cell, or PBMC, population, while the monocytes isolated by magnetic separation account for up to 25 percent of the total PBMC. Monocytes isolated by either method are equally viable. MDDCs differentiated from monocytes purified by both methods were first gated based on DAPI negative or viable cells from total single cells, followed by gating of each cell population.Both isolation methods yielded a low single CD14 positive population and both methods yielded a high total CD11c positive population. Further phenotypic analysis was performed on all the CD11c positive cells and even though magnetic isolation gives a higher percentage of double positive CD11c positive CD14 positive cells, this effect was non-significant when compared with the adherence method. These double positive CD11c positive, CD14 positive cells may be early stage differentiated MDDCs that have not yet dropped the CD14 monocytic marker.When analyzing the single CD11c positive cells, the adherence method gives the highest yield of CD11c positive, CD14 negative cells. These single positive cells may be late stage differentiated MDDCs. Once mastered, the adherence and magnetic isolation techniques can be completed in three to four and one to two hours respectively if they are performed properly.While attempting this procedure, it's important to remember to have fresh dendritic cell differentiation cytokines every 48 hours when the medium is replenished. Working with biohazard fluids such as blood, can be extremely harmful, and the use of appropriate personal protective equipment and disposal methods should always be taken when performing these procedures. This study paves the way for researchers in the field of immunology to explore the use of human monocytes and dendritic cells for therapeutic treatment.After watching this video, you should have a good understanding of how to optimize human monocyte isolation for generating different populations of monocyte derived cells in vitro.

Research

JoVE Journal

Immunology and Infection

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Активация и Измерение NLRP3 Inflammasome деятельности Использование IL-1β в человека моноцитов дендритные клетки

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Поколение человеческих моноцитов дендритных клеток из цельной крови

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Анализ макрофагов Microglia и Monocyte из центральной нервной системы методом проточной цитометрии

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Изоляции и в пробирке культуры мышиных и человека альвеолярные макрофаги

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Характеристика человека Моноцит подмножеств по цельной крови Cytometry анализ потока

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Количественная оценка моноцитной хемотаксической активности in Vivo и характеристика кровяных моноцитов, полученных макрофагами

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

siRNA Электропорация модулировать аутофагии в вирусе простого герпеса типа 1-инфицированных моноцитов производные дендритные клетки

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Поляризация клеток M1 и M2 человека моноцитов и анализ с цитометрией потока при микобактериальной туберкулезной инфекции

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Визуализация воспалительных каспаз, индуцированных близостью в макрофагах, полученных из моноцитов человека

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Определение иммуногенности вакцины с использованием дендритных клеток, полученных из моноцитов крупного рогатого скота

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...