Name: generation of human monocyte-derived dendritic cells from whole blood
Uploaded: 2016-12-24T14:00:00
Description: Watch this Scientific Journal Video about Generation of Human Monocyte-derived Dendritic Cells from Whole Blood at JoVE.com

We would like to thank our technician Karolin Thurnes, Divison of Hygiene and Medical Microbiology, and Dr. Annelies M&#252;hlbacher and Dr. Paul H&#246;rtnagl, Central Institute for Blood Transfusion and Immunological Department, for their valuable help and support regarding this manuscript. We thank the Austrian Science Fund for supporting this work (P24598 to DW, P25389 to WP).

Etikk uttalelse: Skriftlig informert samtykke ble innhentet fra alle deltakende blodgivere ved Sentralinstitutt for blodoverføring og Immunologisk Institutt, Innsbruck, Østerrike. Bruk av anonymiserte left prøver for vitenskapelige formål ble godkjent av etikkomiteen ved Medical University of Innsbruck. 1. Anrikning av perifere mononukleære blodceller (PBMC) <ol><li> Konsentrasjon av PBMC ved sentrifugering. <ol><li> Åpne blod-pakken og delt blod i sterile 50 ml sentrifugerør i henhold ...

<ol>
	<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>Steinman, R. M., Hemmi, H. <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:+translating+innate+to+adaptive+immunity.">Dendritic cells: translating innate to adaptive immunity.</a> Curr Top Microbiol Immunol. 311, 17-58 (2006).</li><li>Steinman, R. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+levels+of+HIV-1+infection+in+cutaneous+dendritic+cells+promote+extensive+viral+replication+upon+binding+to+memory+CD4++T+cells.">Low levels of HIV-1 infection in cutaneous dendritic cells promote extensive viral replication upon binding to memory CD4+ T cells.</a> J Exp Med. 182, 2045-2056 (1995).</li><li>McDonald, D., 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=Recruitment+of+HIV+and+its+receptors+to+dendritic+cell-T+cell+junctions.">Recruitment of HIV and its receptors to dendritic cell-T cell junctions.</a> Science. 300, 1295-1297 (2003).</li><li>Pruenster, 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=C-type+lectin-independent+interaction+of+complement+opsonized+HIV+with+monocyte-derived+dendritic+cells.">C-type lectin-independent interaction of complement opsonized HIV with monocyte-derived dendritic cells.</a> Eur J Immunol. 35, 2691-2698 (2005).</li><li>Wilflingseder, D., 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=IgG+opsonization+of+HIV+impedes+provirus+formation+in+and+infection+of+dendritic+cells+and+subsequent+long-term+transfer+to+T+cells.">IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells.</a> J Immunol. 178, 7840-7848 (2007).</li><li>Kapsenberg, M. L. <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+control+of+pathogen-driven+T-cell+polarization.">Dendritic-cell control of pathogen-driven T-cell polarization.</a> Nat Rev Immunol. 3, 984-993 (2003).</li><li>Mills, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR-dependent+T+cell+activation+in+autoimmunity.">TLR-dependent T cell activation in autoimmunity.</a> Nat Rev Immunol. 11, 807-822 (2011).</li><li>Banki, Z., 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=Complement+as+an+endogenous+adjuvant+for+dendritic+cell-mediated+induction+of+retrovirus-specific+CTLs.">Complement as an endogenous adjuvant for dendritic cell-mediated induction of retrovirus-specific CTLs.</a> PLoS Pathog. 6, e1000891 (2010).</li><li>Posch, W., 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=Antibodies+attenuate+the+capacity+of+dendritic+cells+to+stimulate+HIV-specific+cytotoxic+T+lymphocytes.">Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes.</a> J Allergy Clin Immunol. 130, 1368-1374 (2012).</li><li>Posch, W., 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=Complement-Opsonized+HIV-1+Overcomes+Restriction+in+Dendritic+Cells.">Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells.</a> PLoS Pathog. 11, e1005005 (2015).</li><li>Wilflingseder, D., 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=Immediate+T-Helper+17+Polarization+Upon+Triggering+CD11b/c+on+HIV-Exposed+Dendritic+Cells.">Immediate T-Helper 17 Polarization Upon Triggering CD11b/c on HIV-Exposed Dendritic Cells.</a> J Infect Dis. 212, 44-56 (2015).</li><li>Grassi, 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=Monocyte-derived+dendritic+cells+have+a+phenotype+comparable+to+that+of+dermal+dendritic+cells+and+display+ultrastructural+granules+distinct+from+Birbeck+granules.">Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules.</a> J Leukoc Biol. 64, 484-493 (1998).</li></ol>

Denne protokollen beskriver dannelsen av monocytt-avledede dendrittceller (MDDCs) gjennom isoleringen av humane monocytter fra antikoagulert blod ved hjelp av et magnetisk nanopartikkel-baserte analysen. I denne protokollen blir sentrifugeringstrinn utføres oppstrøms cellen isolasjonsprosedyren, noe som fører til en anrikning av PBMC fraksjon. Selv om celler er mistet under sentrifugeringen, for å overlappe innholdet i en hel blod pakke ved densitetsgradient medium ville kreve 200-300 ml av densitetsgradient medium ...

Dendrittiske celler (DCS) er de viktigste spesialiserte antigenpresenterende celler i immunforsvaret vårt. Umodne DC (IDCS) bor i huden eller i slimhinnene vev og er derfor blant de første immunceller til å samhandle med invaderende patogener. DC representerer bro mellom det medfødte og det adaptive immunsystemet 1, siden de kan aktivere T- og B-celle responser etter deteksjon av patogen. Videre bidrar de til pro-inflammatoriske immunrespons på grunn av sekresjon av høye mengder av cytokiner, slik som IL-1β, IL-6 og IL-12. DCs aktiverer også NK-celler og tiltrekke seg andre immunceller til stedet for infeksjon av chemotaxis. DC kan deles inn i umodne dendrittceller (IDCS) og modne dendrittiske celler (mDCs) 2 basert på deres morfologi og funksjon. Etter erkjennelsen av fremmede antigener ved en av de mange mønstergjenkjenning reseptorer (f.eks toll-like reseptorene, C-typelektiner, reseptorer eller komplement) rikelig uttrykt på celleoverflaten, IDCS gjennomgå store forandringer og begynner å modnes. Under denne modningsprosess, er reseptorer for antigenerobring nedregulert, mens molekyler avgjørende for antigen presentasjon er oppregulert tre. Moden DCs up-regulere vevstypeantigen I og II (MHC I og II), co-stimulerende molekyler som CD80 og CD86, som er avgjørende for antigen presentasjon og aktivering av T-lymfocytter. I tillegg er ekspresjonen av kjemokinreseptoren CCR7 på celleoverflaten induseres, som muliggjør vandring av DC fra perifere vev til lymfeknutene. Migrasjonen er tilrettelagt av å &quot;rulle&quot; av DCs langs en chemokin ligand 19 (CCL19 / MIP-3b) og chemokin ligand 21 (CCL21 / SLC) gradient til lymfeknuter 4-6. Etter migrasjon, mDCs presentere behandlet antigen til naive CD4 + og CD8 + T-celler, og dermed iniforhandle en adaptiv immunrespons mot invaderende patogen 7. Denne interaksjonen med T-celler i lymfeknutene er også forbundet med spredningen av viruset 8. Andre in vitro studier viste at DC effektivt fange og overføre HIV til T-celler, og at denne overføringen resulterer i en kraftig infeksjon 9-12. Disse eksperimentene markere at in vivo HIV utnytter DC som skyttelbussene fra periferien til lymfeknutene. Under antigen presentasjon, DCs utsondre nøkkel interleukiner som former differensiering av effektor T-hjelpeceller, og derfor er utfallet av hele immunrespons mot mikroben fastsatt i dette samspillet. Bortsett fra type 1 (Th1) og type 2 (Th2) effektor T-celler, andre undergrupper av CD4 + T-hjelpeceller (f.eks, type 17 (Th17) og type 22 (Th22) T-celler) har blitt beskrevet, og deres induksjon og funksjonen har blitt grundig undersøkt. DC er dessuten involvert igenerering av regulatoriske T-celler (tregs) 13,14. Disse cellene er immunundertrykkende og kan stoppe eller ned-regulere induksjon eller proliferasjon av effektor-T-celler og er således avgjørende for utvikling av immunitet og toleranse. Menneskekonvensjonelle DC (CDC) omfatter flere undergrupper av celler med en myeloid opprinnelse (dvs. Langerhans-celler (LCS) og dermal og interstitiell DCs) eller en lymfoid opprinnelse (dvs. plasmacytoid DC (PDCs)). For in vitro eksperimenter eller DC vaksinasjonsstrategier, er moncyttavledede DC rutinemessig brukt som modell for dermal DC. Disse cellene viser likheter i fysiologi, morfologi, og funksjonen til vanlige myeloide dendrittiske celler. De er dannet ved tilsetning av interleukin 4 (IL-4) og granulocytt-makrofag-kolonistimulerende faktor (GM-CSF) til monocytter ble isolert fra friske donorer 12,15-18. Dendrittiske celler kan også bli direkte isolert fra dermal eller slimhinne biopsier, eller kan til og med be utviklet seg fra CD34 + hematopoetiske stamceller isolert fra navlestreng blodprøver innhentet ex utero. Her viser vi hvordan monocytter blir isolert og stimulert fra anti-koagulert humant blod etter perifert blod mononukleære celler (PBMC) anrikning av tetthetsgradient-sentrifugering. Etter inkubasjon i 5 dager, humane monocytter under spesielle forhold er differensiert i IDCS og er klar for eksperimentelle prosedyrer i en ikke-klinisk setting.

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			<td>APC Mouse Anti-Human CD19&#160; Clone&#160; HIB19</td>
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			<td>Density gradient media: Ficoll-Paque Premium</td>
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			<td>Dulbecco&#8217;s Phosphate Buffered Saline (D-PBS)</td>
			<td>Sigma-Aldrich</td>
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			<td>Falcon 50mL High Clarity PP Centrifuge Tube</td>
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			<td>Tonbo biosciences</td>
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			<td>Heat Inactivated FBS (Fetal Bovine Serum), EU Approved Origin (South America)</td>
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			<td>PE Mouse Anti-Human CD14&#160; Clone&#160; M5E2</td>
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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.

Etter sentrifugering av anti-koagulert blod ved hjelp av en sukrose pute, er perifere mononukleære blodceller (PBMC) anriket i en interfase på toppen av tettshetsgradient medium (figur 1). Når PBMC-er trukket av, FACS-analyse utført for å karakterisere de forskjellige cellepopulasjoner i løpet av de PBMC ved hjelp avstamning markører (f.eks CD3 for T-lymfocytter, CD14 for monocytter, og CD19 på B-lymfocytter). Figur 2A viser resultatene ...

Watch this Scientific Journal Video about Generation of Human Monocyte-derived Dendritic Cells from Whole Blood at JoVE.com

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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.

Generering av menneske moncyttavledede dendrittiske celler fra Whole Blood

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

The overall goal of this CD14 magnetic beads separation technique is to isolate monocytes from the blood for their further differentiation into dendritic cells or macrophages in vitro. This method can help answer key questions in the field of immunology about the recognition, processing, and presentation of pathogens or antigens by dendritic cells. The main advantage of this isolation procedure is that the 98 to 99%pure monocyte population can be obtained.Demonstrating the procedure will be Karolin Thurnes, a technician from our work group. Begin by splitting the blood pack volume among sterile 50 milliliter centrifuge tubes according to the amount of blood received. Adjust the final volume in each tube to 50 milliliters with sterile Dulbecco's PBS as necessary and centrifuge the samples.Using a sterile one milliliter serological pipette connected to a laboratory vacuum pump, remove the plasma and platelet layers leaving the boundary layers undisturbed. Then, use a new 25 milliliter pipette to combine the mononuclear cell boundary layers from two of the tubes in the new sterile 50 milliliter conical tube. Next, add 15 milliliters of density gradient medium to each of 450 milliliter conical tubes and pipetting slowly and carefully, overlay 25 milliliters of pooled blood cells onto the density gradient in each tube.Separate the cells by centrifugation. Then aspirate the plasma and platelet upper layers and transfer the PBMC interphases into new individual 50 milliliter conical tubes. Wash the PBMCs two times with up to 50 milliliters of sterile DPBS, pulling the cells after the first centrifugation and resuspending the pellet in 50 milliliters of fresh DPBS after the second.After counting, collect the cells by centrifugation for monocyte magnetic particle isolation. To purify the monocytes by magnetic particle isolation, adjust the PBMC concentration to eight times 10 to the 7th cells per milliliter of isolation buffer. Next, vortex the anti-human CD14 magnetic particles thoroughly and mix 50 microliters of particles for every one times 10 to the 7th PBMCs into the cells, and transfer the cell bead solution to a sterile 15 milliliter tube.After 30 minutes at room temperature, adjust the total volume to 12 milliliters of isolation buffer, and transfer two milliliters of the cell particle suspension into each of six sterile round bottom tubes. Immediately place the tubes onto the cell separation magnet for 10 minutes at room temperature. Then remove the supernatant containing the peripheral blood lymphocytes from each tube.Remove the tubes from the magnet and add two milliliters of fresh isolation buffer to each cell sample. Gently pipette up and down to resuspend the cells and particles, and return the tubes to the magnet for another five minutes. At the end of the incubation, remove the supernatant again, and resuspend the cells in two milliliters of fresh isolation buffer as just demonstrated.Then pull the CD14 positive monocyte containing cell fractions into a new 50 milliliter tube and bring the final volume of the tube up to 50 milliliters in sterile DPBS. To differentiate the purified monocytes into dendritic cells, collect the cells by centrifugaiton and resuspend the pellet in 50 milliliters of DPBS. After counting, spin down the cells again and resuspend the pellet at a one times 10 to the 6th cells per milliliter concentration and prewarmed culture medium.Transfer three milliliters of cells into each well of a six well plate and add recombinant human Interleukin four and GMCSF to each well for a 48 hour incubation at 37 degrees Celsius and 5%carbon dioxide. On the second day of culture, restimulate the cells with fresh cytokines and return the plate to the incubator. On day five, pull the supernatants and gently pipette fresh medium into the wells several times to harvest the immature dendritic cells.After density gradient separation, a typical PBMC fraction is comprised of about 4%B-lymphocytes, 28%monocytes and 68%other cells. 44%of which are t-lymphocytes. After magnetic particle separation, flow cytometric analysis of the negative fraction reveals a similar percentage of b-lymphocytes, a higher percentage of the other cell populations and a greatly reduced percentage of monocytes.Characterization of the positive fraction demonstrates the high purificaiton efficiency with over 99%of the cells expressing CD14. After five days of Interleukin four and GMCSF simulation, the CD14 positive cells differentiate into immature CD83 negative DC-SIGN positive monocyte derived dendritic cells which are comparable to dermal dendritic cells in the morphology, behavior, and receptor expression. Once mastered, this technique can be completed in four to five hours if it is performed properly.After watching this video, you should have a good understading of how to isolate monocytes from whole blood and differentiate them into dendritic cells. This technique is very important for researchers in the field of immunology to explore not only dendritic cell, but also monocyte and macrophage function in the primary in vitro human cell system. Following this procedure, other methods like microscopic analysis, flow cytometry, infection or antigen presentation essays can be performed to answer questions about dendritic cell biology, function and interactions with antigens.Don't forget that working with blood can be hazardous and that precautions such as wearing gloves, a lab coat or having a hepatitis vaccination should always be taken before beginning this procedure.

Research

JoVE Journal

Immunology and Infection

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolering av myeloid dendrittiske celler og Epitelceller fra Menneskelig Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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

Aktivisering og måling av NLRP3 Inflammasome aktivitet Bruke IL-1β i Human Monocyte-avledet dendrittiske 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 Alloantigen-specific T Cells from Peripheral Blood

Generation of Human alloantigen spesifikke T-celler fra perifert blod

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

Kultur av makrofag-kolonistimulerende faktor Differensiert Humant Monocyte makrofager

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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

Karakterisering av menneske moncyttavledede dendrittiske celler ved Imaging flowcytometrisystemer: En sammenligning mellom to Monocyte Isolation protokoller

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

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

Dekstran forbedrer Lentiviral signaltransduksjon effektiviteten av Murine og menneskelig primære NK celler

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Generasjon av Discriminative menneskelige monoklonale antistoffer fra sjeldne Antigen-spesifikke B celler i blodet

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

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

siRNA electroporation til modulere Autofagi i herpes simplex virus type 1-infiserte monocytt-avledet dendrittiske celler

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

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

Visualisering av inflammatoriske caspaser indusert nærhet i humane monocytt-avledede 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

Bestemmelse av vaksineimmunogenisitet ved bruk av dendrittiske celler fra bovint monocytt

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