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

Generellt studier humanblod har granskats och godkänts av Institutional Review Board (IRB) av FIU, IRB protokoll godkännande # IRB-13-0440. Mänskliga leukopaks köptes från samhället blodbank i Miami, FL. 1. Isolering av PBMC från Standard densitetsgradient Technique <ol><li> Utföra en 1: 1 spädning av blod med 1x-fosfatbuffrad saltlösning i en T75-flaska. </li><li> Pipett 15 ml av densitetsgradient lösning i 50 ml centrifugrör och noggrant skikt (25 - 30 ml / rör) av det utsp?...

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den aktuella studien bygger på de kända svårigheterna att isolera och generera MDDCs från humant blod, som syftar till att ge en omfattande jämförelse av två väletablerade metoder för generering av MDDCs. Den första metoden jämfört är en känd traditionell metod för att generera MDDCs genom att utnyttja förmågan hos monocyter att vidhäfta till glas eller plast (vidhäftning metod) 21,22,27. Vidhäftningen Metoden är snabb och kostnadseffektiv, och inte kräver användning av komplicerad utru...

Dendritiska celler (DC) är viktiga mediatorer för de medfödda och adaptiva immunsystem. De fungerar att inducera primära immunsvar och underlätta utvecklingen av immunologiskt minne. Dessa celler är primärt ansvariga för antigeninfångnings, migration och T-cellstimulering och därför hänvisas till som professionella antigenpresenterande celler (APC) en .Manipulation av DC: er skulle kunna användas inom ett stort antal olika forskningsområden och inom klinisk miljö för att behandla olika inflammatoriska sjukdomar såsom hiv 6,7, cancer 8, autoimmuna sjukdomar 9, och allergiska reaktioner 10. DCs används också för substansmissbruk forskning för att lösa okända mekanismer och vägar såsom de som förknippas med alkoholberoende 11-14, narkotikamissbruk 13,15, och kombinationen av HIV-infektion och missbruk 16-19. Dessa pågående studier och framtida studier forskning jagn området immunologi gör in vitro generation av DCs extremt viktigt för forskning. Emellertid finns det flera svårigheter som är förknippade med isolering av DC: er från humant blod som de bara utgör 0,1-1% av de totala mononukleära blodceller 20. Hittills några av de väl etablerade metoder för generering av DC: er in vitro består av plast eller glas vidhäftning av monocyter 21,22, densitetsgradientcentrifugering 23, specifik markör baserad separation, såsom magnetisk aktiverad cellsortering 22, fluorescensaktiverad cellsortering 24 positiv selektion av CD14 + monocyter använder dextran-belagda magnetiska nanopartiklar 25, och snabb isolering av höggradigt renade monocyter med fullt automatiserad negativ cellselektion 26. Men den bästa metoden för val förblir kontroversiell. Därför, för att förbättra DC generationens teknik, har flera metoder utvecklats i vilkarenheten hos dessa celler kan ökas avsevärt genom differentiering från renade CD34 + progenitorceller och monocyter isolerade från perifert blod mononukleära celler (PBMC) 27. Såsom nämnts tidigare, är en allmänt använd och populär metod för att generera som härstammar från monocyter dendritiska celler (MDDCs) för att undersöka förmågan hos monocyter att vidhäfta till glas eller plast (vidhäftning metod) 21,22,27. Vidhäftningen metoden är en snabb och enkel metod som inte kräver användning av komplicerad utrustning. Men några nackdelar med denna process inkluderar lymfocyter föroreningar, låg flexibilitet, och monocyt gående manipulation 28. En alternativ metod för att vidhäftningen metod är den magnetiska isolering av monocyter från de totala PBMC, särskilt med användning av en human monocyt anrikning kit, som är utformad för att isolera monocyter från PBMC genom negativ selektion 26. Under denna procedur, är oönskade celler riktade bort med tetramerantikroppskomplex och dextran-belagda magnetiska partiklar. Fördelen med denna isoleringsmetoden är att de oönskade märkta celler separeras med användning av en magnet medan målceller kan fritt hälldes av in i ett nytt rör utan behov av kolumner. Hittills med tillgängligheten av specifika monoklonala antikroppar som kan märka unika cellpopulationer har den magnetiska separationstekniken blir inte bara ytterligare en metod, utan en nödvändighet för isolering av sällsynta celler inom immunologi. Till exempel, tekniker såsom magnetisk cellsortering med kommersiellt tillgängliga paramagnetiska MACS-nanopartiklar har underlättat utvecklingen av nya metoder för forskning och kliniska tillämpningar 22,29. Dessutom har de senaste studier som jämför DC generationen från monocytvidhäftning och Mac teknikmetoder visade en högre DC renhet och livskraft med hjälp av MACS separerade monocyter 22,30. Den aktuella studien presenteraren jämförelse mellan två metoder för generering av humana DCs från monocyter isolerade från PBMC: 1) monocyt isolering genom vidhäftning och 2) monocyt isolering genom negativ selektion med användning av en kommersiell mänsklig monocyt anrikning kit. Denna studie ger belägg för att den negativa selektions magnetiska separationsförfarande för att isolera monocyter genererar den högsta avkastningen av monocyter med inga signifikanta skillnader i monocyt livsduglighet jämfört med monocyter isolerade genom vidhäftning metod. I sin tur, efter sju dagar, monocyterna som isolerats genom magnetisk separation differentieras till MDDCs med signifikant högre proliferativ kapacitet och större mängd celler som uttrycker dubbla positiva (CD11c + / CD14 +) fenotyp utan att påverka MDDC viabilitet. Sammantaget skiljer den aktuella studien från tidigare studier som refereras ovan, eftersom det visar förmågan hos båda teknikerna samtidigt generera monocyter som kan tillväxa och differentiera till CD11c +MDDCs (&gt; 70%) efter sju dagar i kultur utan att kompromissa deras livskraft. Dessutom ger den nuvarande strategin för första gången karakterisering av olika CD11c / CD14 MDDCs populationer av avbildning flödescytometri. Sammanfattningsvis, eftersom DCs spelar en central roll när det gäller forskning inom immunologi, olika parametrar måste beaktas när man överväger hur de kommer och vilka metoder som används för att isolera och kultur dem in vitro. Därför syftar denna studie för att ge insikter om två olika metoder för monocyt isolering och hur dessa metoder differentiellt påverka monocyt lönsamhet och avkastning på sikt påverkar dendritiska celler livskraft, tillväxt, och fenotyp. Dessa fynd kommer att bidra mycket till området immunologi och kommer att ge ett detaljerat protokoll av DC isolering, rening, och karakterisering.

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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> Monocyt Utbyte av magnetisk separation är högre jämfört med monocyt Yield genom vidhäftning metod Data som presenteras i figur 1 display PBMC och monocyt kroppar av trypanblått uteslutningsmetoden på dagen för isolering av PBMC och separation av monocyter. I genomsnitt, monocyter isolerade av följsamhet metoden svarade för cirka 6,2 procent av den totala PBMC medan monocyter is...

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.

Karakterisering av mänskliga monocythärledda dendritiska celler från Imaging Flow Cytometry: en jämförelse mellan två monocyt Isoleringsprotokoll

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

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Immunology and Infection

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

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1ÃƒÅ½Ã‚Â² in Human Monocyte-derived Dendritic Cells

Aktivering och Mätning av NLRP3 inflamma aktivitet med användning av IL-1β i human monocyt-härledda dendritiska celler

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

Generation av mänskliga monocythärledda dendritiska celler från helblod

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

Analys av Microglia och monocyt-härledda makrofager från det centrala nervsystemet genom flödescytometri

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

Isolering och In Vitro kultur av murina och mänskliga alveolära makrofager

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

Karakterisering av mänskliga monocyt delmängder av helblod Flow flödescytometri analys

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

Kvantifiering av Monocytisk Chemotaktisk aktivitet in vivo och karakterisering av Blodmonocyte härledda makrofager

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 elektroporation att modulera autofagi i herpes simplex virus typ 1-infekterade Monocyte-derived dendritiska celler

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

Polarisering av M1- och M2-humana monocytbaserade celler och analys med flödescytometri vid mykobakterie tuberculosisinfektion

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

Visualisering av inflammatorisk kaspas inducerad närhet i humana monocyt-härledda makrofager

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

Bestämning av vaccinimmunogenicitet med hjälp av dendritiska celler härledda från nötkreatur

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