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.

Genel insan kanı çalışmaları gözden geçirmiş ve IRB protokol onay # IRB-13-0440 Mali İstihbarat Birimi Kurumsal İnceleme Kurulu (KİK) tarafından onaylanmıştır. İnsan leukopaklar Miami, FL topluluk kan bankasından satın alınmıştır. Standart Yoğunluk Gradient Tekniği ile PBMC&#39;ler 1. İzolasyonu <ol><li> T75 şişesi içinde 1x-fosfat tamponlu tuzlu su ile kanı 1 seyreltme: 1 gerçekleştirin. </li><li> Pipet 15 ila 50 ml santrifüj tüpleri içine yoğunluk gradyan solüsyonu ve dikkatli bir şekilde katman - Bu gradyan seyreltilmiş kan (25 30 mL / tüp). </li><li> 1 hızlanma ve 0 yavaşlama ile 1200 x g&#39;de 20 dakika süreyle santrifüjlenir. </li><li> Santrifüj işleminden sonra, yeni bir 50 ml&#39;lik santrifüj tüpüne arabirim katmanı (beyaz kan hücreleri) toplamak ve PBS içinde iki kere (1.080 x g&#39;de 3 dakika) ardından hücreler yıkanır. Her zaman süpernatant atın. </li><li> Amonyum klorid, potasyum lize (ACK) tampon maddesi ile hücreleri tedavi (kırmızı kan hücreleri lize etmek için). tampon ve 10 ml4 ° C&#39;de 15 dakika boyunca cubate. </li><li> 1.080 x g&#39;de 3 dakika boyunca, PBS ile iki kez santrifüj hücre yıkayın ve her bir zaman supernatant atın. </li><li> PBMC&#39;ler içerecek olan pelet kaydedin. </li><li> tercih edilen monosit saflaştırma yöntemi ile devam edin. </li></ol> Bağlılık Yöntemiyle 2. Monosit Arıtma <ol><li> L-glutamin (300 mg / ml) ihtiva eden tam bir hücre kültür ortamı hazırlayın penisilin (50 U / ml) -streptomycin (50 ug / ml) ve% 10 cenin sığır serumu ile takviye edilmiştir. </li><li> PBMC&#39;ler nemlendirilmiş bir kuluçka makinesi içinde 37 ° C&#39;de ve% 5 CO2 seviyesinde tam ortam, 10 ml başına 5 x 10 7 hücre konsantrasyonunda T75 şişesi içinde kültürlenmesi ile yaklaşık 2 saat boyunca yapışmaya izin verin. </li><li> İnkübasyondan sonra, kültür şişesi yapışmayan kayan hücreleri çıkarmak ve yavaşça PBS ile iki kez yapışmış olan hücrelerin yıkayın. </li><li> insan granülosit-Makr 2 ul / ml ile desteklenmiş tam hücre kültür ortamı içinde yapışkan hücreleri inkübeophage koloni uyarıcı faktör (GM-CSF) ve interlökin 4 (IL-4), 10 ug / ml&#39;lik bir stok konsantrasyonu C&#39;de saklandı. </li><li> orta yarısı değiştirin ve sitokinler her 48 saat doldurmak. </li><li> MDDCs monositlerin farklılaşması için 7 gün - 5 bekleyin. </li></ol> Manyetik Ayırma Yöntemiyle 3. Monosit Arıtma <ol><li> 5 ml&#39;lik bir polistiren tüp içine dökün ve hücre / ml&#39;lik bir konsantrasyonda PBS tamponu içinde askıya tekrar standart yoğunluk gradyan tekniği PBMC&#39;ler toplayın. </li><li> 50 ul / ml hücreleri kullanılarak insan monosit zenginleştirme kokteyl ekleyin karıştırın ve 10 dakika boyunca 4 ° C&#39;de inkübe edin. </li><li> İnkübasyondan sonra, 50 ul / ml hücreleri kullanılarak manyetik parçacıkların ilave edilir, iyice karıştırın ve 5 dakika boyunca 4 ° C&#39;de inkübe edin. </li><li> PBS tampon çözeltisi ilave edilerek 2.5 ml&#39;lik toplam hacme kadar hücre süspansiyonu getirmek aşağı 2 ve yukarı pipetlenerek iyice karıştırın - 3 kez. </li><li> Manyetik cihazın içine bir kapak olmadan polistiren tüp yerleştirin ve 2 RT&#39;de kenara.5 dakika. </li><li> inkübasyondan sonra ve bir sürekli hareket halinde, mıknatıs pick up ve temiz bir 50 ml&#39;lik santrifüj tüpüne istenen saflaştırılmış monosit fraksiyonu dökün. </li><li> Tüm hücre kültürü insan granülosit-makrofaj koloni stimüle edici faktör (GM-CSF) 2 ul / ml ile takviye edilmiş ortam ve interlökin 4 yapışık hücreleri inkübe (IL-4), 10 ug / ml&#39;lik bir stok konsantrasyonu C&#39;de saklandı. </li><li> Her 48 saat, orta yarısı değiştirin 5 dakika boyunca 1.080 xg&#39;de aşağı spin ve yeni medya (cRPMI) ve sitokinler ile pelet yeniden askıya. </li><li> MDDCs monositlerin farklılaşması için 7 gün - 5 bekleyin. </li></ol> 4. Tripan Mavi Dışlama Canlılık Testi Not: PBMC, monositler ve MDDCs hücre verimini ve canlılığı elde etmek için bu yöntem kullanınız. <ol><li> Standart tripsin-EDTA kullanılarak ve matara ve PBS kullanarak hücre pelet yıkar gerçekleştirmek Hasat hücreleri. pelet boyutuna bağlı olarak, yenidenpelet çözmek için PBS 5 ila 10 ml askıya. </li><li> Seyreltilmiş hücre peleti ile tripan mavisi reaktif 1 seyreltme: mikro-santrifüj tüpünde, 1 gerçekleştirin. </li><li> Bu karışımı, bir hücre içine kısım 10 ul slayt sayarak ve üreticinin protokolüne uygun olarak, otomatik bir hücre sayacı sonuçları okuyun. otomatik bir hücre sayacı mevcut değilse, benzer hücre sayısı, bir hemasitometre veya manuel sayacı kullanılarak mümkündür. </li></ol> 5. MTT Testi <ol><li> Bir 96-çukurlu plaka içindeki her birine tam ortam 4 x 10 4/100 ul&#39;lik bir konsantrasyonda plakası hücreleri. Hücreler kültüre uyum için 24 saat izin verin. </li><li> Inkübe ve sırasıyla 0 (Yok gün 0), 36 (3 gün) ve 84 (gün 7) saatte okumaları gerçekleştirin. </li><li> İstenilen kuluçka tamamlanmasından sonra, ortamını çıkarın ve tek başına 100 ul PBS ile değiştirin. </li><li> Hazırlama 3- (4,5-dimetiltiazol-2-il) dimetilpropil 10 ml ekleyerek -2,5-difenil tetrazolyum bromür çözeltisi (MTT) (5 mg / ml)sodyum dodesil sülfat, 1 g (SDS) il sülfoksit (DMSO). </li><li> Her bir oyuğa MTT çözeltisi 10 ul (5 mg / ml) ilave edin ve 2 saat daha 37 ° C&#39;de inkübe edin. </li><li> inkübasyondan sonra, PBS ve dönüştürülmemiş MTT karışımı (sarı renkli çözelti) ihtiva süpernatantlar kaldırmak. </li><li> Her bir oyuğa, SDS-DMSO çözeltisi 100 ul ilave edin ve bir saat daha inkübe edilir. </li><li> Bir mikrolevha okuyucu kullanılarak 540 nm&#39;de absorbansı okumak. </li></ol> 6. Hücre Yüzey Görüntüleme Sitometrisi ile Boyama Analizi <ol><li> Saflaştınldı monositlerden farklılaşma 7 gün sonra, 1.5 ml tüpler uygun sayıya monosit türevli dendritik hücrelerin 1x10 6 Hücre numuneleri dağıtın. </li><li> ısıyla inaktive 50 ul (HI), insan serumu kullanılarak bloke hücreler ile hücre yüzeye lekelemesi başlatın, 10 dakika boyunca 4 ° C&#39;de inkübe edin. </li><li> İnkübasyondan sonra, santrifüj hücreler 5 dakika için 720 x g hızında, süpernatant atılır. </li><li> pelle hücret, ilgili florokrom-eşlenik antikorlar (örneğin, anti-CD11c, anti-CD14 ya da) ekleyin ve ışıktan koruyarak 20 dakika boyunca 4 ° C&#39;de inkübe edin. tazminat için gerekli beri ayrı tüplerde) tek florokrom lekeli örnekler / ml hazırlamak. </li><li> İnkübasyondan sonra, 5 dakika boyunca 720 x g&#39;de PBS tamponu ve santrifüj 1 ml her seferinde iki kez yıkama hücreleri. </li><li> ışıktan korunan hücreleri tutulması, akış sitometri analizi için bir hücre / 100 ul bir konsantrasyonda PBS tamponu içinde yeniden askıya. </li><li> canlı hücreler üzerinde kapısı için, önce, imalatçının talimatlarına uygun olarak alet sitometrisi tek hücre görüntüleme akış hücreleri elde etmek Her tüpe DAPI 1 ul ekle. </li></ol> 7. İstatistiksel Analiz <ol><li> Girdi bir e-tablodaki tüm verileri. </li><li> Öğrencinin t-testi ve / veya tek yön uygun olarak ANOVA kullanarak sonuçları karşılaştırın. </li><li> p ise &lt;0.05 istatistiksel olarak anlamlı farklılıklar göz önünde bulundurun. </li&gt; </ol>

<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>

All authors disclose no competing financial interests.

izole edilmesi ve insan kanından MDDCs üretilmesi bilinen zorlukların göre, bu çalışmada MDDCs üretilmesi için iki köklü yöntemlerin bir karşılaştırma sağlamak için amaçlanmıştır. Kıyasla ilk yöntem, cam veya plastik (yapışma yöntemi) 21,22,27 uymaları monositlerin yeteneği yararlanarak MDDCs üretilmesi için iyi bilinen bir geleneksel bir yöntemdir. yapışma kullanım olup, etkili, hızlı ve düşük maliyetli ve karmaşık ekipman kullanımını gerektirmez. Ancak, bu sürec...

Dendritik hücreler (DC), doğal ve adaptif bağışıklık sistemlerinin temel aracılarıdır. Onlar primer immün yanıtları teşvik ve immünolojik hafıza gelişimini kolaylaştırmak için çalışır. Bu hücreler, antijen tutma, taşıma ve T hücre uyarılması için esas olarak sorumlu olan ve bu nedenle, DClerin .Manipulation farklı tedavi etmek için araştırma alanları çok çeşitli boyunca ve klinik ortamda yararlanılabilir 1 profesyonel antijen sunan hücreler (APC&#39;ler) olarak adlandırılır HIV 6,7, kanser 8 gibi enflamatuvar hastalıklar, oto-bağışıklık hastalıkları 9 ve alerjik tepkiler 10. DH&#39;ler ayrıca literatürde alkol bağımlılığı 11-14, ilaç bağımlılığı 13,15 ve HIV enfeksiyonu ve madde kötüye kullanımı, 16-19 kombinasyonu ile ilişkili bilinmeyen ve oluşum mekanizmaları çözmek için Madde Araştırma için kullanılmaktadır. Bu devam eden çalışmalar ve gelecekteki araştırma çalışmaları in immünoloji saha araştırması için son derece önemli DC&#39;ler in vitro nesil olun. Toplam kan tek çekirdekli hücreleri 20% 1 - Bununla birlikte, bir 0,1 teşkil insan kanından DH&#39;lerin izole edilmesine ile ilişkili çeşitli zorluklar bulunmaktadır. Bugüne kadar, DH&#39;lerin in vitro üretilmesi için iyi bilinen yöntemlerin bazıları, 22 ayırma manyetik aktif hücre, floresan aktive hücre sınıflandırma Monositler 21,22 plastik ya da cam yapışması, yoğunluk gradyanlı santrifüj 23, spesifik bir işaretleyici göre ayrılması oluşmaktadır 24, dekstran-kaplı manyetik nanopartiküller 25 ve tam otomatik negatif hücre seçimi 26 ile yüksek oranda saflaştırılmış monositlerin hızlı izolasyon kullanılarak CD14 + monositler pozitif seçim. Ancak, seçim en iyi yöntemi tartışmalıdır. Bu nedenle, DC oluşturma teknikleri geliştirmek için, çeşitli yöntemler hangi geliştirilmiştirBu hücrelerin saflığı önemli ölçüde, periferal kan tek-çekirdekli hücreleri (PBMC), 27 ile izole edilmiş, saflaştırılmış CD34 + projenitör hücreleri ve monositler farklılaşmanın arttırılabilir. Önce belirtildiği gibi, monosit türevli dendritik hücreler (MDDCs) üretmek için yaygın olarak kullanılan bir ve popüler bir yöntem cam veya plastik (yapışma yöntemi) 21,22,27 uymaları monositlerin yeteneğini keşfetmek olduğunu. yapışma yöntemi, karmaşık ekipman kullanımının gerektirmeyen, hızlı ve basit bir yöntemdir. Ancak, bu sürecin bazı dezavantajları lenfosit kirlenme, düşük esneklik ve monosit geçici manipülasyon 28 arasındadır. Yapışma yönteme alternatif bir yöntem, özel olarak negatif seçim 26 tarafından PBMC&#39;lerden monositleri izole etmek için tasarlanmış olan bir insan monosit zenginleştirme kiti kullanılarak toplam PBMC&#39;lerden monositler manyetik izolasyondur. Bu işlem sırasında, istenmeyen hücrelere tetramerik ile çıkarılması için hedeflenirantikor komplekslerinden ayrılmakta ve dekstran-kaplı manyetik parçacıklar. Bu izolasyon yönteminin avantajı, istenmeyen etiketli hücreler hedef hücreler serbest sütun gerek olmadan yeni bir tüp içine boşaltılır edilebilir ise bir mıknatıs kullanılarak ayrıştırılmasıdır. Bugüne kadar, benzersiz bir hücre popülasyonu etiket spesifik monoklonal antikorların mevcudiyeti, manyetik ayırma tekniği ek bir yöntem, ancak immünoloji alanında az hücrelerin izolasyonu için bir zorunluluk değildir, sadece olmuştur. Örneğin, manyetik hücre gibi teknikler araştırma ve klinik uygulamaları 22,29 için yeni yaklaşımların gelişmesine olanak sağlamıştır piyasada mevcut paramanyetik MACS-nanopartiküller ile sıralama. Ayrıca, monosit bağlılık ve MACS teknoloji yöntemleri DC nesil karşılaştıran son araştırmalar monositler 22,30 ayrılmış MACS kullanarak daha yüksek bir DC saflık ve canlılığı ortaya koymuştur. Bu çalışma sunarticari bir insan monosit zenginleştirme kiti kullanılarak yapışma 1), monosit izolasyonu ve 2), monosit izolasyon negatif seçim kriteri: PBMC&#39;lerden izole monositlerde insan DH&#39;lerinin üretimi için iki yöntem arasındaki karşılaştırması. Bu çalışma, yapışma yöntemiyle izole edilmiş monositler ile karşılaştırıldığında monositlerin izole etmek için negatif seçim manyetik ayırma işlemi monosit yaşam kabiliyetinde önemli bir farklılıklar ile monositlerin yüksek verim oluşturur dair kanıt sağlar. Buna karşılık, yedi gün sonra, manyetik ayırma ile izole monositler MDDC canlılığı etkilemeden anlamlı olarak daha yüksek çoğalma kapasitesi ve çift pozitif (CD11c + / CD14 +) fenotip sentezleyen hücrelerin yüksek miktarda MDDCs ayrışmıştır. aynı anda çoğalan ve CD11c ayırma yöntemi edebilen monositleri üretmek için her iki tekniğin becerisi gösterir çünkü genel olarak, bu çalışma + Yukarıda atıfta bulunulan önceki çalışmalardan farklıyaşama yetenekleri tehlikeye sokulmadan kültür içinde yedi gün sonra MDDCs (&gt;% 70). Buna ek olarak, mevcut yaklaşım sitometri görüntüleme akışı ile farklı CD11c / CD14 MDDCs popülasyonlarının ilk kez karakterizasyonu sağlar. DC&#39;ler immünoloji alanında araştırma ile ilgili bir odak noktası rolü oynamaya beri elde edilir ve hangi yöntemleri in vitro onları izole ve kültür için nasıl kullanıldığını göz önüne alındığında Özetle, farklı parametreler dikkate alınmalıdır. Bu nedenle, bu çalışmada iki farklı monosit izolasyon yöntemleri ve nasıl bu yöntemler farklı olarak sonunda dendritik hücre canlılığı, proliferasyon ve fenotip etkileyen monosit canlılığını ve verimini etkileyen anlayışlar sağlamayı amaçlamaktadır. Bu bulgular immünoloji alanında büyük katkı sağlayacaktır ve DC izolasyonu, saflaştırılması ve karakterizasyonu ayrıntılı bir protokol sağlayacaktır.

<table><tbody><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td>17-5442-03</td><td>Must be used at RT</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>ACK Lysing buffer</td><td>Quality Biological</td><td>118-156-101</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Life Technologies</td><td>22400-089</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240-062</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>RoboSep buffer</td><td>StemCell</td><td>20104</td><td></td></tr><tr><td>EasySep Human monocyte enrichment kit</td><td>StemCell</td><td>19059</td><td></td></tr><tr><td>TC20 Automated cell counter</td><td>Bio-Rad</td><td>145-0101</td><td></td></tr><tr><td>FITC-Dextran</td><td>Sigma Aldrich</td><td>FD4-100MG</td><td></td></tr><tr><td>Trypan blue stain (0.4%)</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>Synergy 2 multi-mode reader</td><td>Biotek</td><td>7131000</td><td></td></tr><tr><td>XTT Sodium salt bioreagent (XTT)</td><td>Sigma Aldrich</td><td>X4626-100MG</td><td></td></tr><tr><td>Dimethyl sulfoxide bioreagent (DMS)</td><td>Sigma Aldrich</td><td>D8418-500ML</td><td></td></tr><tr><td>Thiazolyl blue tetrazolium bromide (MTT)</td><td>Sigma Aldrich</td><td>m5655-500MG</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Bio-Rad</td><td>161-0302</td><td></td></tr><tr><td>Phenazine Methosulfate (DMSO)</td><td>Sigma Aldrich</td><td>P9626-1G</td><td></td></tr><tr><td>Inactivated (HI) Human Serum</td><td>Chemicon</td><td>S1-100ML</td><td></td></tr><tr><td>Accuri C6 Flow Cytometer</td><td>BD Accuri</td><td>653119</td><td></td></tr><tr><td>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> Manyetik Ayırma ile Monosit Verim bağlılık Yöntemi ile Monosit Verimi Higher karşılaştırıldığında edilir PBMC&#39;ler ve monositlerin ayrılması izolasyon gün tripan mavisiyle çıkarma metodu kullanılarak Şekil 1 ekran PBMC ve monosit hücre sayımı sunulan veriler. manyetik ayırma ile izole monositler PBMC kadar yüzde 25 paya sahip olması ortalama bağlılık yönt...

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.

Görüntüleme Sitometrisi ile İnsan Monosit türetilmiş dendritik hücrelerle Karakterizasyonu: İki Monosit İzolasyon Protokoller Arasında Bir Karşılaştırma

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

characterization-human-monocyte-derived-dendritic-cells-imaging-flow

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.4K Görüntüleme.  Florida International University. 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.

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

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Bir<em&gt; In vitro</emİnsan Makrofaj Farklılaşma ve Polarizasyon heterojenliği Eğitim için&gt; Model

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

İmmünoloji ve Enfeksiyon

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

İnsan Çift Gradient Santrifüj ile Monositlerinde ve teflon kaplı Hücre Kültürü Çantalar Makrofagların Onların Farklılaşma İzolasyonu

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

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

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Akış Sitometresi ile Merkezi Sinir Sisteminden Mikroglia ve Monosit Türevi Makrofajların Analizi

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

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

Miktar monosit hicret ve köpük hücre oluşumu kronik inflamatuar koşullara sahip bireylerden

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

İnsan monosit alt kümeleri tam kan tarafından karakterizasyonu Akış Sitometresi Analizi

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

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

Diseke İmplant Dokularının İmmün Fonksiyon Analizi için Yüksek Boyutluluk Akış Sitometrisi

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Biyomühendislik

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Birincil İnsan Monositlerinin İzolasyon, Transfeksiyon ve Kültürü

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

Sialidaz Tedavisi Kullanılarak İzole PBMC'lerden De-Sialillenmiş Monosit Türetilmiş DC'ler

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Yazar Spotlight: Hücre Yüzeyinin Sialidaz Bazlı Enzimatik İşlemi Kullanılarak Dendritik Hücrelerin Olgunlaşması 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Farklı Sialillenmiş Fenotiplere Sahip Monosit Kaynaklı Dendritik Hücrelerin Üretimi

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

Görüntüleme Sitometrisi ile İnsan Monosit türetilmiş dendritik hücrelerle Karakterizasyonu: İki Monosit İzolasyon Protokoller Arasında Bir Karşılaştırma

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Bir

İnsan Çift Gradient Santrifüj ile Monositlerinde ve teflon kaplı Hücre Kültürü Çantalar Makrofagların Onların Farklılaşma İzolasyonu

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

Akış Sitometresi ile Merkezi Sinir Sisteminden Mikroglia ve Monosit Türevi Makrofajların Analizi

Miktar monosit hicret ve köpük hücre oluşumu kronik inflamatuar koşullara sahip bireylerden

İnsan monosit alt kümeleri tam kan tarafından karakterizasyonu Akış Sitometresi Analizi

Diseke İmplant Dokularının İmmün Fonksiyon Analizi için Yüksek Boyutluluk Akış Sitometrisi

Birincil İnsan Monositlerinin İzolasyon, Transfeksiyon ve Kültürü

Farklı Sialillenmiş Fenotiplere Sahip Monosit Kaynaklı Dendritik Hücrelerin Üretimi

Farklı Sialillenmiş Fenotiplere Sahip Monosit Kaynaklı Dendritik Hücrelerin Üretimi