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).

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 ster...

<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. 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>

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.

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			<td>APC Mouse Anti-Human CD19&#160; Clone&#160; HIB19</td>
			<td>BD Biosciences</td>
			<td>555415</td>
			<td></td>
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			<td>BD Biosciences</td>
			<td>551073</td>
			<td></td>
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			<td>BD Imag Anti-Human CD14 Magnetic Particles&#160;</td>
			<td>BD Biosciences</td>
			<td>557769</td>
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			<td>BD Imagnet</td>
			<td>BD Biosciences</td>
			<td>552311</td>
			<td></td>
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			<td>BSA (Albumin Fraction V)</td>
			<td>Carl Roth</td>
			<td>EG-Nr 2923225</td>
			<td></td>
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			<td>Costar 6 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Costar</td>
			<td>3506</td>
			<td></td>
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			<td>Density gradient media: Ficoll-Paque Premium</td>
			<td>GE Healthcare Bio-Sciences</td>
			<td>17-5442-03</td>
			<td></td>
		</tr>
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			<td>Dulbecco&#8217;s Phosphate Buffered Saline (D-PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
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			<td>Falcon 10mL Serological Pipet</td>
			<td>Corning</td>
			<td>357551</td>
			<td></td>
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			<td>Falcon 25mL Serological Pipet</td>
			<td>Corning</td>
			<td>357525</td>
			<td></td>
		</tr>
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			<td>Falcon 50mL High Clarity PP Centrifuge Tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
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			<td>Falcon Round-Bottom Tubes</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
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			<td>FITC Mouse Anti-Human CD3&#160; Clone&#160; HIT3a</td>
			<td>BD Biosciences</td>
			<td>555339</td>
			<td></td>
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			<td>Ghost Dye Violet 510 (Cell Viability Reagent)</td>
			<td>Tonbo biosciences</td>
			<td>13-0870</td>
			<td></td>
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			<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>
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			<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>
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			<td>Microcentrifuge tubes, 1,5 ml, SuperSpin</td>
			<td>VWR</td>
			<td>211-0015</td>
			<td></td>
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			<td>PE Mouse Anti-Human CD14&#160; Clone&#160; M5E2</td>
			<td>BD Biosciences</td>
			<td>555398</td>
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			<td>PE Mouse Anti-Human CD209&#160; Clone&#160; DCN46</td>
			<td>BD Biosciences</td>
			<td>551265</td>
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			<td>RPMI-1640 medium</td>
			<td>Sigma-Aldrich</td>
			<td>R0883</td>
			<td></td>
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			<td>UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></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.

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 ...

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.

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

Research

JoVE Journal

Immunology and Infection

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 ...

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

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

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

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 regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

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

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

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