Name: generation of human monocyte-derived dendritic cells from whole blood
Uploaded: 2016-12-24T14:00:00.000+00:00
Duration: 7 min 35 s
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 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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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. 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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 therefore cannot be considered cost efficient. Additionally, the enrichment of PBMCs by centrifugation results in a cleaner cell population, which positively influences the attachment of the anti-human CD14 magnetic particles during the isolation process. Magnetic-nanoparticle isolation of monocytes results in a viable monocyte population, which can be further used for in vitro differentiation to various DC subsets or macrophages.
Advantages of this technique compared to other protocols used in the past, like adherence-mediated purification on tissue culture or gelatin-coated plastic dishes, are the high purity and homogeneity of the isolated cell population, as well as the convenience and speed. In general, it is important to work quickly but thoroughly when working with cells in order to avoid prolonged exposure to non-physiological conditions. The anti-human CD14 magnetic particles used in this protocol are optimized for the positive selection or depletion of CD14-bearing leukocytes; therefore, the magnetic nanoparticles bind directly to the cells of interest and are not removed after the procedure. This could be an issue that researchers must address before certain experiments or analyses are performed, since results could be influenced or altered by cell-bound magnetic nanoparticles. Alternatively, negative selection assays are available, which are designed to deplete unwanted cells and to keep the cells of interest untouched. For the generation of MDDCs, the anti-human CD14 magnetic nanoparticles used to isolate monocytes present no complication because, during the differentiation into MDDCs, cytokine-stimulated monocytes down-regulate the CD14 receptor. Thus, the magnetic nanoparticles are removed without further elution or detaching procedures, and the MDDCs are tested negative for CD14 receptor expression by flow cytometry. Since culture medium containing FCS is used during the differentiation in this protocol, MDDCs obtained here are for research use only. For MDDCs in clinical settings, special media without the need of serum have been developed. In addition, autologous plasma can be collected after the first centrifugation step (step 1.1.4), heat-inactivated, and used in the culture medium. Besides autologous plasma, commercially-available human plasma can also be added to the culture medium to avoid possible background reactivity in long-term assays due to xenogeneic proteins in FCS.
Alternatively, dendritic cells can be directly isolated from dermal or mucosal biopsies or can be developed from CD34+ hematopoietic progenitor cells isolated from umbilical cord blood samples obtained ex utero.
Nonetheless, MDDCs are the most widely-used model for dendritic cells, since the direct isolation of DCs from biopsies is more complex to perform. It can also lead to inefficient cell numbers that often vary due to differences in the donor quality. Similar problems can occur when DCs are generated from CD34+ stem cells isolated from cord blood.
For future applications, inducible pluripotent stem cells (iPSCs) could be used instead of CD14-magnetic bead isolation of monocytes from blood. The advantages of using iPSCs are that not only DC subsets, but all hematopoietic cells of interest, can be generated from one donor (T cells, B cells, and NK cells) and that patient-specific iPSCs can be used to characterize the interactions of autologous immune cells in a personalized manner.
The most critical step during the isolation of monocytes is the density gradient centrifugation step, since accurate separation of the red and white blood cells is only achieved when the brake of the centrifuge is turned off. This determines the outcome of the down-stream isolation efficiency.
In conclusion, this protocol is a very fast, efficient, and cost-effective way to generate a viable and homogenous monocyte-derived dendritic cell population through the isolation of human monocytes from anti-coagulated blood.

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><tbody><tr><td>APC Mouse Anti-Human CD19 Clone HIB19</td><td>BD Biosciences</td><td>555415</td><td></td></tr><tr><td>APC Mouse Anti-Human CD83 Clone HB15e</td><td>BD Biosciences</td><td>551073</td><td></td></tr><tr><td>BD Imag Anti-Human CD14 Magnetic Particles</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&amp;rsquo;s Phosphate Buffered Saline (D-PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Falcon 10&amp;nbsp;ml Serological Pipet</td><td>Corning</td><td>357551</td><td></td></tr><tr><td>Falcon 25&amp;nbsp;ml Serological Pipet</td><td>Corning</td><td>357525</td><td></td></tr><tr><td>Falcon 50&amp;nbsp;ml 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 Clone 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)</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 Clone M5E2</td><td>BD Biosciences</td><td>555398</td><td></td></tr><tr><td>PE Mouse Anti-Human CD209 Clone 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 0.5 M 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 representative FACS analysis of PBMCs collected and stained after density gradient centrifugation. Out of the gated population (Figure 2A, upper plot), we detected 4.03% B lymphocytes, 28.20% monocytes, and 67.62% other cells (Figure 2A, middle plot), of which 43.62% were T lymphocytes (Figure 2A, lower plot).
PBMCs are then incubated with CD14 magnetic particles and, following incubation on a magnet, the negative fraction containing the peripheral blood lymphocytes (PBLs) is analyzed by FACS using the selection of abovementioned lineage markers. Compared to PBMCs, we measured similar percentages of B lymphocytes (4.65%, Figure 2B, middle plot), higher percentages of other cells (88.61%, Figure 2B, middle plot), and strongly reduced percentages of monocytes (6.59%, Figure 2B, middle plot). Characterization of the positive fraction (Figure 3) demonstrated a high separation efficiency, since the purity of the isolated cells is over 97% (Figure 3, left plot), of which 99.56% (Figure 3, right plot) expressed high levels of CD14 on the cell surface.
IL4 and GM-CSF stimulation of monocytes for 5 days results in differentiation into monocyte-derived dendritic cells, which are comparable to dermal dendritic cells in morphology, behavior, and receptor expression19. FACS analysis of monocyte-derived dendritic cells on day 5 shows a homologous population (Figure 4, left plot) expressing high levels of CD11b, CD11c (not shown), and the C-type lectin DC-SIGN. No expression of the DC maturation marker CD83 is detected (Figure 4, right plot), and cells also lose the monocyte marker CD14 (not shown).
<img alt="Figure 2" src="/files/ftp_upload/54968/54968fig1.jpg" /> 
Figure 1: Separation of blood components by density gradient centrifugation. PBMCs are enriched in an interphase on top of the separation medium by density gradient centrifugation. Layers before (left) and after (right) centrifugation are shown. <a href="http://cloudflare.jove.com/files/ftp_upload/54968/54968fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54968/54968fig2.jpg" /> 
Figure 2: Flow cytometric analyses of PBMCs (A) and PBLs (B). (A) PBMCs are harvested, stained for lineage markers (mouse-anti-human CD3, CD14, and CD19 antibodies), and analyzed by flow cytometry. Dot plots of a representative result are shown. (B) The negative fraction containing PBLs is characterized by lineage markers (mouse-anti-human CD3, CD14, and CD19 antibodies) and analyzed by flow cytometry. Dot plots of a representative result are shown. <a href="http://cloudflare.jove.com/files/ftp_upload/54968/54968fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54968/54968fig3.jpg" /> 
Figure 3: Flow cytometric analyses of monocytes. The isolated monocytes (positive fraction) are characterized using lineage markers (mouse-anti-human CD3, CD14, and CD19 antibodies) and analyzed by flow cytometry. Dot plots of a representative result are shown.
<img alt="Figure 4" src="/files/ftp_upload/54968/54968fig4.jpg" /> 
Figure 4: Flow cytometric analyses of monocyte-derived dendritic cells. Monocytes are stimulated for 5 days with IL4 and GM-CSF and stained for characteristic DC markers, like CD11b, CD11c (not shown), the C-type lectin DC-SIGN, and the maturation marker CD83. Dot plots of a representative result are shown.

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 ...

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Nanyang Technological University

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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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biology

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

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 ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

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 ...

De-Sialylated Monocyte-Derived-DCs from Isolated PBMCs Using Sialidase Treatment

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

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

Author Spotlight: Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface 

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

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

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

Source: Sim, W. J. et al., <a href="https://app.jove.com/v/54128">Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes.</a>&#160;J. Vis. Exp.&#160;(2016).
This video demonstrates the generation of immature, mature, and tolerogenic dendritic cells, or DCs, from human monocytes. The incubation of monocytes with growth factors and cytokines leads to the production of immature DCs. In response to immune regulatory molecules, immature DCs differentiate into tolerogenic and mature DCs.

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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