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).

Ethics statement: Written informed consent was obtained from all participating blood donors by the Central Institute for Blood Transfusion &#38; Immunological Department, Innsbruck, Austria. The use of anonymized leftover specimens for scientific purposes was approved by the Ethics Committee of the Medical University of Innsbruck.
1. Enrichment of Peripheral Blood Mononuclear Cells (PBMCs)
<ol>
	<li>Concentration of PBMCs by centrifugation.
	<ol>
		<li>Open the blood pack and split blood in sterile 50 ml centrifuge tubes according to the amount of blood received.</li>
		<li>If necessary, adjust the volume to 50 ml for each tube with sterile Dulbecco&#39;s Phosphate-Buffered Saline (D-PBS).</li>
		<li>Place the tubes into a centrifuge and spin them at 400 x g for 15 min at room temperature (RT) without brake.</li>
		<li>Draw off the upper layer containing plasma and platelets using a sterile 10 ml serological pipette, leaving the boundary layer undisturbed. 
		&#8203;NOTE: If autologous plasma instead of FCS is used to supplement the culture medium, the plasma must be collected and heat-inactivated in this step.</li>
		<li>Collect the mononuclear cells (boundary layers) from two 50 ml centrifuge tubes and transfer them into one sterile 50 ml tube using a sterile 10 ml serological pipette.</li>
		<li>Adjust the volume to 50 ml for each tube using sterile D-PBS.</li>
		<li>Place the tubes into a centrifuge and spin them at 400 x g for 15 min at RT without brake.</li>
		<li>Draw off the upper layer containing the plasma and platelets using a sterile 10 ml serological pipette, leaving the boundary layer undisturbed.</li>
		<li>Collect the mononuclear cells (boundary layers) from the 50 ml centrifuge tubes and transfer them into two sterile 50 ml collection tubes using a sterile 10 ml serological pipette.</li>
		<li>Adjust the volume to 50 ml for each tube with sterile D-PBS.</li>
	</ol>
	</li>
	<li>Separation of PBMCs by density gradient centrifugation (Figure 1).
	<ol>
		<li>Prepare four 50 ml tubes and pipette 15 ml of density gradient medium into each tube.</li>
		<li>Take 25 ml of pooled cells/blood from the collection tube (step 1.1.10) and carefully overlay the density gradient medium.</li>
		<li>Place the tubes into a centrifuge and spin them at 700 x g for 30 min at RT without brake.</li>
	</ol>
	</li>
	<li>Collection and purification of PBMCs.
	<ol>
		<li>Draw off the upper layer containing the plasma and platelets using a sterile 10 ml serological pipette, leaving the PBMC layer undisturbed.</li>
		<li>Collect the PBMC interphase from all tubes and pool the cells in two sterile 50 ml tubes using a sterile 25 ml serological pipette.</li>
		<li>Adjust the volume to 50 ml for each tube with sterile D-PBS. Place the tubes into a centrifuge and spin them at 400 x g for 15 min at 4 &#176;C with the brake.</li>
		<li>Aspirate the supernatant and re-suspend the cells in sterile D-PBS. Pool the cells from both tubes and adjust the volume to 50 ml with sterile D-PBS.</li>
		<li>Place the tubes into the centrifuge and spin them at 400 x g for 10 min at 4 &#176;C with brake.</li>
		<li>Aspirate the supernatant and re-suspend the cells in sterile D-PBS. Adjust the volume to 50 ml with sterile D-PBS and pipette 50 &#181;l to a 1.5 ml tube containing 450 &#181;l of D-PBS.</li>
		<li>Vortex the 1.5 ml tube vigorously and count the cells in a Neubauer chamber or any other cell counting instrument.</li>
		<li>Place the 50 ml tubes into the centrifuge and spin them at 400 x g for 10 min at 4 &#176;C with brake.</li>
	</ol>
	</li>
</ol>
2. Isolation of Monocytes by Anti-human CD14 Magnetic Particles
<ol>
	<li>Labeling of PBMCs with magnetic particles.
	<ol>
		<li>Adjust the PBMC concentration to 8 x 107 cells/ml using isolation buffer.</li>
		<li>Vortex the anti-human CD14 magnetic particles thoroughly and add 50 &#181;l of particles for every 1 x 107 PBMCs.</li>
		<li>Mix the cell-particle suspension thoroughly and incubate it at RT for 30 min.</li>
	</ol>
	</li>
	<li>Separation of the positive and negative fractions.
	<ol>
		<li>Adjust the volume up to 12 ml using isolation buffer.</li>
		<li>Prepare six sterile round-bottom tubes and pipette 2 ml of the cell-particle suspension into each tube.</li>
		<li>Immediately place the tubes on the magnet. Incubate them for 10 min at RT.</li>
		<li>Keep the tubes on the magnet and carefully draw off the supernatant. The supernatant contains the negative fraction.</li>
		<li>Remove the tube from the magnet and add 2 ml of isolation buffer.</li>
		<li>Gently re-suspend the cells by pipetting up and down.</li>
		<li>Place the tubes back on the magnet. Incubate for 5 min at RT.</li>
		<li>Keep the tubes on the magnet and carefully draw off the supernatant. The supernatant contains the negative fraction.</li>
		<li>Remove the tubes from the magnet and add 2 ml of isolation buffer to each tube. Gently re-suspend the cells by pipetting up and down.</li>
		<li>Transfer the positive fraction to a sterile 50 ml tube and adjust the volume to 50 ml using sterile D-PBS.</li>
	</ol>
	</li>
</ol>
3. Stimulation of Isolated Monocytes with IL-4 and GM-CSF
<ol>
	<li>Place the tube into the centrifuge and spin it at 400 x g for 10 min at 4 &#176;C with brake.</li>
	<li>Aspirate the supernatant and re-suspend the cells in 50 ml of D-PBS.</li>
	<li>Pipette 50 &#181;l into a 1.5 ml tube containing 450 &#181;l of D-PBS. Vortex the 1.5 ml tube vigorously and count the cells in a Neubauer chamber or another cell counting instrument.</li>
	<li>Place the 50 ml tube into the centrifuge and spin it at 400 x g for 10 min at 4 &#176;C with brake. 
	NOTE: If the negative fraction, containing peripheral blood lymphocytes (PBLs), is also required, perform washing and counting steps similar to those of the positive fraction (steps 3.1-3.5).</li>
	<li>Adjust the cell concentration to 1 x 106/ml with pre-warmed culture medium (RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 1% L-Glutamine solution) and pipette 3 ml/well into 6-well plates. 
	NOTE: If using heat-inactivated autologous plasma, replace the 10% FCS with 1-5% autologous plasma, according to the experimental set-up.</li>
	<li>Stimulate the monocytes with recombinant human IL-4 (250 U/ml) and recombinant human GM-CSF (1,000 U/ml) by pipetting the cytokines directly into the culture medium. Incubate at 37 &#176;C and 5% CO2.</li>
	<li>Re-stimulate the cells with IL-4 (250 U/ml) and GM-CSF (1000 U/ml) after 48 hr by pipetting the cytokines directly into the culture medium. 
	NOTE: If there are any signs of acidification shown by the pH indicator in the medium, replace half of the medium with pre-warmed culture medium.</li>
	<li>Harvest immature dendritic cells on day 5 for down-stream experiments by pipetting the cultured cells out of the 6-well plate and into a 50 ml centrifuge tube after pipetting the culture medium up and down a few times.</li>
	<li>Count the cells and stain a sample of isolated monocytes with anti-human antibodies against CD11b, CD11c, and DC-SIGN/CD209, together with a cell viability dye. To avoid unspecific binding of the antibodies during surface staining, the use of Fc receptor blocking reagents or bovine serum albumin (BSA) buffers is highly recommended.</li>
	<li>Analyze the sample using the cell viability dye, according to the manufacturer&#39;s protocol, by performing flow cytometry to assess the purity and viability of the generated iDCs.</li>
</ol>

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	<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. M., Inaba, K., Turley, S., Pierre, P., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+capture,+processing,+and+presentation+by+dendritic+cells:+recent+cell+biological+studies.">Antigen capture, processing, and presentation by dendritic cells: recent cell biological studies.</a> Hum Immunol. 60, 562-567 (1999).</li><li>Schwarz, J., Sixt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+Dendritic+Cell+Haptotaxis.">Quantitative Analysis of Dendritic Cell Haptotaxis.</a> Methods Enzymol. 570, 567-581 (2016).</li><li>Weber, M., Sixt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+chemotactic+dendritic+cell+migration+in+explanted+mouse+ear+preparations.">Live cell imaging of chemotactic dendritic cell migration in explanted mouse ear preparations.</a> Methods Mol Biol. 1013, 215-226 (2013).</li><li>Sixt, M., Lammermann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+analysis+of+chemotactic+leukocyte+migration+in+3D+environments.">In vitro analysis of chemotactic leukocyte migration in 3D environments.</a> Methods Mol Biol. 769, 149-165 (2011).</li><li>Randolph, G. J., Angeli, V., Swartz, 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=Dendritic-cell+trafficking+to+lymph+nodes+through+lymphatic+vessels.">Dendritic-cell trafficking to lymph nodes through lymphatic vessels.</a> Nat Rev Immunol. 5, 617-628 (2005).</li><li>Wilflingseder, D., Banki, Z., Dierich, M. P., Stoiber, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+promoting+dendritic+cell-mediated+transmission+of+HIV.">Mechanisms promoting dendritic cell-mediated transmission of HIV.</a> Mol Immunol. 42, 229-237 (2005).</li><li>Pope, M., Gezelter, S., Gallo, N., Hoffman, L., 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=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>

The authors have nothing to disclose.

This protocol describes the generation of monocyte-derived dendritic cells (MDDCs) through the isolation of human monocytes from anti-coagulated blood using a magnetic nanoparticle-based assay. In this protocol, the centrifugation steps are performed upstream the cell isolation procedure, which leads to an enrichment of the PBMC fraction. Although cells are lost during centrifugation, to overlay the content of a whole blood pack on density gradient medium would require 200-300 ml of the density gradient medium and theref...

Dendritic cells (DCs) are the most important specialized antigen-presenting cells of our immune system. Immature DCs (iDCs) reside in the skin or in mucosal tissues and are therefore among the first immune cells to interact with invading pathogens. DCs represent the bridge between the innate and the adaptive immune system1, since they can activate T- and B-cell responses following pathogen detection. Furthermore, they contribute to pro-inflammatory immune responses because of the secretion of high amounts of cytokines, such as IL-1&#946;, IL-6, and IL-12. DCs also activate NK cells and attract other immune cells to the site of infection by chemotaxis.
DCs can be divided into immature dendritic cells (iDCs) and mature dendritic cells (mDCs)2 based on their morphology and function. After the recognition of foreign antigens by one of the many pattern recognition receptors (e.g., toll-like receptors, C-type lectins, or complement receptors) abundantly expressed on the cell surface, iDCs undergo major changes and start to mature. During this maturation process, receptors for antigen capture are down-regulated, whereas molecules essential for antigen presentation are up-regulated3. Mature DCs up-regulate the major histocompatibility complexes I and II (MHC I and II), co-stimulatory molecules like CD80 and CD86, which are essential for antigen presentation and activation of T-lymphocytes. Additionally, the expression of chemokine receptor CCR7 on the cell surface is induced, which enables the migration of DCs from peripheral tissues to the lymph nodes. The migration is facilitated by the &#34;rolling&#34; of DCs along a chemokine ligand 19 (CCL19/MIP-3b) and chemokine ligand 21 (CCL21/SLC) gradient to the lymph nodes4-6.
Following migration, mDCs present the processed antigen to na&#239;ve CD4+ and CD8+ T cells, thus initiating an adaptive immune response against the invading pathogen7. This interaction with T cells in the lymph nodes is also associated with the spread of the virus8. Other in vitro studies revealed that DCs efficiently capture and transfer HIV to T cells and that this transmission results in a vigorous infection9-12. These experiments highlight that in vivo HIV exploits DCs as shuttles from the periphery to the lymph nodes. During antigen presentation, DCs secrete key interleukins that shape the differentiation of effector T helper cells, and therefore, the outcome of the entire immune response against the microbe is determined at this very interaction. Apart from type 1 (Th1) and type 2 (Th2) effector T cells, other subsets of CD4+ T helper cells (e.g., type 17 (Th17) and type 22 (Th22) T cells) have been described, and their induction and function have been investigated thoroughly. DCs are furthermore involved in the generation of regulatory T cells (Tregs)13,14. These cells are immunosuppressive and can stop or down-regulate induction or proliferation of effector T cells and are thus crucial for developing immunity and tolerance.
Human conventional DCs (cDCs) comprise several subsets of cells with a myeloid origin (i.e., Langerhans Cells (LCs) and dermal and interstitial DCs) or a lymphoid origin (i.e., plasmacytoid DCs (pDCs)). For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used as a model for dermal DCs. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by the addition of interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to monocytes isolated from healthy donors12,15-18. Dendritic cells can also be directly isolated from dermal or mucosal biopsies, or can even be developed from CD34+ hematopoietic progenitor cells isolated from umbilical cord blood samples obtained ex utero. 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. After incubation for 5 days, human monocytes under specific conditions are differentiated into iDCs and are ready for experimental procedures in a non-clinical setting.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>APC Mouse Anti-Human CD19&#160; Clone&#160; HIB19</td>
			<td>BD Biosciences</td>
			<td>555415</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD83&#160; Clone&#160; HB15e</td>
			<td>BD Biosciences</td>
			<td>551073</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Imag Anti-Human CD14 Magnetic Particles&#160;</td>
			<td>BD Biosciences</td>
			<td>557769</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Imagnet</td>
			<td>BD Biosciences</td>
			<td>552311</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (Albumin Fraction V)</td>
			<td>Carl Roth</td>
			<td>EG-Nr 2923225</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Costar</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient media: Ficoll-Paque Premium</td>
			<td>GE Healthcare Bio-Sciences</td>
			<td>17-5442-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (D-PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10mL Serological Pipet</td>
			<td>Corning</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 25mL Serological Pipet</td>
			<td>Corning</td>
			<td>357525</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL High Clarity PP Centrifuge Tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Tubes</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD3&#160; Clone&#160; HIT3a</td>
			<td>BD Biosciences</td>
			<td>555339</td>
			<td></td>
		</tr>
		<tr>
			<td>Ghost Dye Violet 510 (Cell Viability Reagent)</td>
			<td>Tonbo biosciences</td>
			<td>13-0870</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-862</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS (Fetal Bovine Serum), EU Approved Origin (South America)</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hettich Rotanta 460R</td>
			<td>Hettich</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-4 CC</td>
			<td>PromoKine</td>
			<td>C-61401</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation buffer: BD IMag Buffer (10X)&#160;</td>
			<td>BD Biosciences</td>
			<td>552362</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 1,5 ml, SuperSpin</td>
			<td>VWR</td>
			<td>211-0015</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD14&#160; Clone&#160; M5E2</td>
			<td>BD Biosciences</td>
			<td>555398</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD209&#160; Clone&#160; DCN46</td>
			<td>BD Biosciences</td>
			<td>551265</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Sigma-Aldrich</td>
			<td>R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

After centrifugation of anti-coagulated blood using a sucrose cushion, peripheral blood mononuclear cells (PBMCs) are enriched in an interphase on top of the density gradient medium (Figure 1). After the PBMCs are drawn off, FACS analysis is performed to characterize the different cell populations within the PBMCs using lineage markers (e.g., CD3 for T lymphocytes, CD14 for monocytes, and CD19 for B lymphocytes). Figure 2A shows the results of a ...

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.

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

generation-of-human-monocyte-derived-dendritic-cells-from-whole-blood

Research

JoVE Journal

Immunology and Infection

20.1K Views.  Medical University of Innsbruck. 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.

Video: Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human 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 presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

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

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

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

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

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

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

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

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.

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.

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

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

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

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

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

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...