This research is supported by the National Institute on Alcohol Abuse and Alcoholism, award K99/R00 AA021264. Additional lab support as part of startup package has been received from the Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, and FIU- Office of Research and Economic Development. Gianna Casteleiro was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Overall human blood studies have been reviewed and approved by the Institutional Review Board (IRB) of FIU, IRB protocol approval # IRB-13-0440. Human leukopaks were purchased from the community blood bank in Miami, FL.
1. Isolation of PBMCs by Standard Density Gradient Technique
<ol>
	<li>Perform a 1:1 dilution of blood with 1x-phosphate-buffered saline in a T75 flask.</li>
	<li>Pipette 15 ml of density gradient solution into 50 ml centrifuge tubes and carefully layer (25 - 30 ml/tube) of the diluted blood over this gradient.</li>
	<li>Centrifuge for 20 min at 1,200 x g with acceleration of 1 and deceleration of 0.</li>
	<li>After centrifugation, collect the interface layer (white blood cells) into a new 50 ml centrifuge tube and wash cells twice in PBS (3 min at 1,080 x g). Discard supernatant each time.</li>
	<li>Treat cells with ammonium-chloride-potassium lysing (ACK) buffer (to lyse red blood cells). Add 10 ml of buffer and incubate for 15 min at 4 &#176;C.</li>
	<li>Wash cells twice with PBS, centrifuge for 3 min at 1,080 x g and discard supernatant each time.</li>
	<li>Save the pellet, which will contain the PBMCs.</li>
	<li>Proceed with monocyte purification method of choice.</li>
</ol>
2. Monocyte Purification by Adherence Method
<ol>
	<li>Prepare complete cell culture medium containing L-glutamine (300 mg/ml) and supplemented with penicillin (50 U/ml)-streptomycin (50 &#181;g/ml) and 10% fetal bovine serum.</li>
	<li>Allow PBMCs to adhere for approximately 2 hr by culturing them in a T75 flask at a concentration of 5 x 107 cells per 10 ml of complete medium at 37 &#176;C and 5% CO2 in a humidified incubator.</li>
	<li>After incubation, remove non-adherent floating cells from culture flask and gently wash adherent cells twice with PBS.</li>
	<li>Incubate adherent cells in complete cell culture medium supplemented with 2 &#181;l/ml of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) stored at a stock concentration of 10 &#181;g/ml.</li>
	<li>Change half of the medium and replenish cytokines every 48 hr.</li>
	<li>Allow 5 - 7 days for the differentiation of monocytes into MDDCs .</li>
</ol>
3. Monocyte Purification by Magnetic Separation Method
<ol>
	<li>Collect PBMCs from standard density gradient technique, pour into a 5 ml polystyrene tube and re-suspend in PBS buffer at a concentration of cells/ml.</li>
	<li>Add human monocyte enrichment cocktail using 50 &#181;l/ml cells, mix well and incubate at 4 &#176;C for 10 min.</li>
	<li>After incubation, add magnetic particles using 50 &#181;l/ml cells, mix well and incubate at 4 &#176;C for 5 min.</li>
	<li>Bring cell suspension up to a total volume of 2.5 ml by adding PBS buffer, mix well by pipetting up and down 2 - 3 times.</li>
	<li>Place the polystyrene tube without a cap into the magnetic device and set aside at RT for 2.5 min.</li>
	<li>After incubation and in one continuous motion, pick up the magnet and pour the desired purified monocyte fraction into a clean 50 ml centrifugation tube.</li>
	<li>Incubate adherent cells in complete cell culture medium supplemented with 2 &#181;l/ml of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) stored at a stock concentration of 10 &#181;g/ml.</li>
	<li>Every 48 hr, change half of the medium, spin down at 1,080 x g for 5 min and re-suspend pellet with new media (CRPMI) and cytokines.</li>
	<li>Allow 5 - 7 days for the differentiation of monocytes into MDDCs.</li>
</ol>
4. Trypan Blue Exclusion Viability Assay
Note: Use this technique to obtain cell yield and viability of PBMCs, monocytes, and MDDCs.
<ol>
	<li>Harvest cells using standard trypsin-EDTA and perform washes of the flasks and the cell pellet using PBS. Depending on the size of the pellet, resuspend in 5 to 10 ml of PBS to dissolve the pellet.</li>
	<li>In a micro-centrifuge tube, perform a 1:1 dilution of trypan blue reagent with diluted cell pellet.</li>
	<li>From this mix, aliquot 10 &#181;l into a cell counting slide and read results using an automated cell counter according to manufacturer&#39;s protocol. If an automated cell counter is not available, a similar cell count is possible using a hemocytometer or a manual counter.</li>
</ol>
5. MTT Assay
<ol>
	<li>Plate cells at a concentration of 4 x 104/100 &#181;l of complete media into each well in a 96-well plate. Allow 24 hr for cells to adjust to culture.</li>
	<li>Incubate and perform readings at 0 (day 0), 36 (day 3) and 84 (day 7) hr respectively.</li>
	<li>At completion of desired incubation, remove media and replace with 100 &#181;l of PBS alone.</li>
	<li>Prepare 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (MTT) (5 mg/ml) by adding 10 ml of dimethyl sulfoxide (DMSO) to 1 g of sodium dodecyl sulfate (SDS).</li>
	<li>Add 10 &#181;l (5 mg/ml) of MTT solution to each well and incubate at 37 &#176;C for 2 additional hours.</li>
	<li>After incubation, remove supernatants which contain PBS and unconverted MTT mixture (yellow-color solution).</li>
	<li>Add 100 &#181;l of SDS-DMSO solution to each well and incubate for one additional hour.</li>
	<li>Read absorbance at 540 nm using a microplate reader.</li>
</ol>
6. Cell Surface Staining Analysis by Imaging Flow Cytometry
<ol>
	<li>After 7 days of differentiation from purified monocytes, dispense 1x106 cell aliquots of the monocyte-derived dendritic cells into the appropriate number of 1.5 ml tubes.</li>
	<li>Start cell surface staining by blocking cells using 50 &#181;l of heat inactivated (HI) human serum, incubate at 4 &#176;C for 10 min.</li>
	<li>After incubation, centrifuge cells at 720 x g for 5 min, discard supernatant.</li>
	<li>To cell pellet, add respective fluorochrome-conjugated antibodies (e.g., anti-CD11c or anti-CD14) and incubate at 4 &#176;C for 20 min protected from light. In separate tubes, prepare single fluorochrome-stained samples/ml) since they are needed for compensation.</li>
	<li>After incubation, wash cells twice in 1 ml of PBS buffer and centrifuge each time at 720 x g for 5 min.</li>
	<li>Keeping cells protected from light, re-suspend in PBS buffer at a concentration of cells/100 &#181;l for flow cytometry analysis.</li>
	<li>In order to gate on viable cells, add 1 &#181;l of DAPI to each tube prior to acquiring the cells on the single cell imaging flow cytometry instrument according to manufacturer&#39;s instructions.</li>
</ol>
7. Statistical Analysis
<ol>
	<li>Input all data in a spreadsheet.</li>
	<li>Compare results using the student&#39;s t-test and/or one-way ANOVA as appropriate.</li>
	<li>Consider differences to be statistically significant if p &#60; 0.05.</li>
</ol>

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All authors disclose no competing financial interests.

Based on the known difficulties of isolating and generating MDDCs from human blood, the present study aimed to provide a comprehensive comparison of two well-established methods for the generation of MDDCs. The first method compared is a well-established traditional method for generating MDDCs by exploiting the ability of monocytes to adhere to glass or plastic (adherence method) 21,22,27. The adherence method is fast and cost effective, and does not require the use of complex equipment. However, some disadvantages of this process include lymphocyte contamination, low flexibility, monocyte transient manipulation 22,30,31. The second alternative method compared is the magnetic isolation of monocytes from total peripheral blood mononuclear cells (PBMCs) using a human monocyte enrichment kit 26, which is designed to isolate monocytes from PBMCs by negative selection. The advantage of this isolation method is that the unwanted cells are labeled and separated using a magnet while the target cells are freely poured off into a new tube without the need of columns and without directly targeting the cells with antibodies. Regardless of monocyte isolation method (adherent vs. magnetic separation), after both procedures, one of the critical steps within the protocol is the in vitro stimulation of monocytes with granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which are required to generate MDDCs. This is a common and well established protocol to generate MDDCs from blood mononuclear cells without compromising antigen capturing and processing capacity characteristic of immature MDDCs 32. One of the major limitations of the techniques commonly used for the in vitro generation of DCs is the low yield of precursor cells such as the monocytes. As reported above, monocytes isolated by the adherence method accounted for approximately 6.2 percent of total PBMCs, while monocytes isolated by magnetic separation accounted for up to 25 percent of PBMCs. According to the literature, ranges for differential white blood cell count in normal adults for monocytes is from 2 - 10% 31 while DCs only constitute 0.1 - 1% of total blood mononuclear cells 20. Therefore, the high yield of monocytes (~ 25%) obtained after magnetic isolation could be due to impurity of cell population and lack of antibody and/or magnetic particle binding to unwanted cells.
To achieve a high yield and purity of the isolated monocytes, all the critical steps in the protocol should be carefully followed. During isolation of PBMCs from blood by standard density gradient centrifugation, there are a few critical steps. First, pipetting and carefully layering of blood over density gradient solution into 50 ml centrifuge tubes is a step that requires practice since pipetting harshly can cause the mixing of this solution and blood, which can lead to a weak PBMC layer that is hard to isolate, or it can result in the complete mixture of red blood cells with PBMCs. In this step, it is important to maintain a constant and relatively slow flow of pipetting to have a well-defined layer of blood and a distinct PBMC layer. Second, centrifugation in the next step with acceleration 1 and deceleration 0 is very critical. If these settings are not used for the first centrifugation step, it will lead to mixing of the blood with the density gradient solution erratically and will not separate the PBMCs from the rest of the blood components. Third, it is important to incubate the PBMCs with Ammonium-Chloride-Potassium (ACK) lysing buffer for no longer than the optimized time (10 - 15 min) since longer incubation with ACK can lead to reduction of viable PBMCs along with red blood cells. In addition, it is also important to carry out the PBS washes after ACK incubation. However, if the red blood cells are not lysed with the first ACK step and they still continue to appear after washes, another round of ACK lysing is highly recommended.
After isolation of white blood cells, there are a few critical steps to keep in mind when performing the adherence method to generate monocytes. First, washing off the floating lymphocytes after two hours of PBMCs incubation is very important since the presence of floating lymphocytes can cause less monocytes to adhere and can also result in lower MDDC purity. Although these washing steps are critical, excessive and harsh washing (more than recommended, i.e., twice) can cause washing off the adherent monocytes too, resulting in lower monocyte yield. Second, incubating the cells with complete medium and cytokines is critical since the cytokines are responsible for differentiation of monocytes into MDDCs. In addition, changing media and replenishing the cytokines every 48 hr is important for the differentiation and survival of the cells. While changing media, it is also crucial to save the floating MDDCs and return them back into culture along with fresh media and cytokines since there are some MDDCs that can detach from the surface during the process of differentiation. It also important to make sure that the monocytes are seeded at the recommended concentration, attached and closely spaced because they need cell to cell contact in order to grow. When performing the magnetic separation method to generate monocytes, there are a few critical steps in order to get a good yield and high purity of monocytes. First, it is critical to maintain the cell concentration recommended by the manufacturer. It is also important to not disturb the polystyrene tube when placed in the magnet, as it may lead to lesser yield and lesser purity. As presented above, this technique leads to a significantly higher monocyte yield (Figure 1) when compared to the adherence method, which may be related to the presence of other cells. Furthermore, when the cells were characterized by flow cytometry, the magnetic method shows a higher percentage of double positive cells (CD11C+/CD14+), which may correlate with the presence of other cells or may also be due to the presence of different stages of MDDC differentiation.
Another important aspect of in vitro MDDC generation regardless of yield and purity is the ability to generate viable and proliferative MDDCs. The current study provides evidence to demonstrate that both purification methods induce proliferation of MDDCs (Figure 3) as shown by the increase in cell metabolic activity during seven days in culture. In addition, the generation of MDDCs from monocytes purified by magnetic separation showed a significantly higher rate of proliferation compare to MDDCs generated from monocytes purified by adhesion (Figure 3). These findings are in accordance with previous literature demonstrating that generation of DCs from CD34+ cells is a proliferative process due to the presence of proliferative DC progenitors in human blood 27,33. However, there are controversial findings demonstrating that monocytes can also differentiate into DCs without any evidence of proliferation 34,35. It is relevant to point out that there is a lot of controversy about the in vitro generation of DCs and regarding whether DCs are generated from proliferating precursors or from differentiation of monocytes. For instance, in vitro production of DC from adherent peripheral blood cells have been shown to also enrich for progenitor cells that are capable of proliferation after exposure to GM-CSF 27 and there is also evidence demonstrating that the yield of DCs derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes 33. Overall, the current study demonstrates an increased in proliferation of monocytic cells in culture that by seven days result in a total MDDCs population with &#62; 70% CD11c+ phenotype (adherence = 72% &#177; 11 versus magnetic = 79% &#177; 19). It is relevant to point out that when further phenotypic analysis was performed, although not significant, the monocytes isolated by the magnetic separation method resulted in MDDCs with a higher double positive phenotype (adherence = 49% &#177; 7 CD11c+/CD14+ versus magnetic = 73% &#177; 15 CD11c+/CD14+) while the monocytes isolated by adhesion resulted in MDDCs with a higher single positive phenotype (adherence = 23% &#177; 7 CD11c+/CD14- versus magnetic = 6% &#177; 7 CD11c+/CD14-). MDDCs with a higher double positive phenotype (CD11c+/CD14+) may be under early stages of differentiation while MDDCs with a higher single positive phenotype may be under late stages of differentiation.
In summary, this study provides evidence to support that the negative selection magnetic separation procedure to isolate monocytes generates the highest yield of monocytes with no significant differences in monocyte viability when compared with monocytes isolated by adherence method. In turn, after seven days, the monocytes isolated by magnetic separation differentiated into MDDCs with significantly higher proliferative capacity and higher amount of cells expressing double positive (CD11c+/CD14+) phenotype without affecting MDDC viability. When comparing both methods, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating (Figure 3) and differentiating (Figure 4) into CD11c+ MDDCs after seven days in culture since both methods yielded &#62; 70% CD11c+ MDDCs. However, further analysis looking at maturation markers may prove useful to fully characterize the functional MDDC population. In conclusion, both methods are advantageous alternatives for the isolation of CD11c+ MDDCs and provide an adequate yield for research applications and may even be useful for future clinical applications. The lab is currently focusing on the generation of MDDCs to study the effects of alcohol abuse and other substances of abuse on the innate immune system, in particularly the ability of these substances to modify MDDC phenotype and function. Therefore, the current study will provide a baseline of MDDC characterization and enhance the ability to proceed with further experimentation in order to elucidate the molecular mechanisms mediating monocyte-derived dendritic cell function in the context of substance abuse.

Dendritic cells (DCs) are essential mediators of the innate and adaptive immune systems. They function to induce primary immune responses and facilitate the development of immunological memory. These cells are primarily responsible for antigen capture, migration and T cell stimulation and are therefore referred to as professional antigen presenting cells (APCs) 1.Manipulation of DCs could be utilized across a wide variety of research fields and in the clinical setting to treat different inflammatory diseases such as HIV 6,7, cancer 8, autoimmune diseases 9, and allergic responses 10. DCs are also being used for substance abuse research in order to solve unknown mechanisms and pathways such as those associated with alcohol dependence 11-14, drug dependence 13,15, and the combination of HIV infection and substance abuse 16-19. These ongoing studies and future research studies in the field of immunology make in vitro generation of DCs extremely important for research. However, there are several difficulties associated with isolating DCs from human blood as they only constitute 0.1 - 1% of total blood mononuclear cells 20.
To date, some of the well-established methods for the generation of DCs in vitro consists of plastic or glass adherence of monocytes 21,22, density gradient centrifugation 23, specific marker based separation such as magnetic activated cell sorting 22, fluorescent activated cell sorting 24, positive selection of CD14+ monocytes using dextran-coated magnetic nanoparticles 25, and rapid isolation of highly purified monocytes using fully automated negative cell selection 26. However, the best method of choice remains controversial. Therefore, to improve DC generation techniques, several methods have been developed in which the purity of these cells can be greatly increased by differentiation from purified CD34+ progenitor cells and monocytes isolated from peripheral blood mononuclear cells (PBMCs) 27. As mentioned prior, a widely used and popular method for generating monocyte derived dendritic cells (MDDCs) is to explore the ability of monocytes to adhere to glass or plastic (adherence method) 21,22,27. The adherence method is a rapid and straightforward method that does not require the use of complex equipment. However, some disadvantages of this process include lymphocyte contamination, low flexibility, and monocyte transient manipulation 28. An alternative method to the adherence method is the magnetic isolation of monocytes from total PBMCs , particularly with the use of a human monocyte enrichment kit, which is designed to isolate monocytes from PBMCs by negative selection 26. During this procedure, unwanted cells are targeted for removal with tetrameric antibody complexes and dextran-coated magnetic particles. The advantage of this isolation method is that the unwanted labeled cells are separated using a magnet while target cells can be freely poured off into a new tube without the need for columns. To date, with the availability of specific monoclonal antibodies that can label unique cell populations, the magnetic separation technique has become not simply an additional method, but a necessity for the isolation of rare cells in the field of immunology. For instance, techniques such as magnetic cell sorting with commercially available paramagnetic MACS-nanoparticles have facilitated the development of new approaches for research and clinical applications 22,29. Furthermore, recent research studies comparing DC generation from monocyte adherence and MACS technology methods have demonstrated a higher DC purity and viability using MACS separated monocytes 22,30.
The current study presents a comparison between two methods for the generation of human DCs from monocytes isolated from PBMCs: 1) monocyte isolation by adherence and 2) monocyte isolation by negative selection using a commercial human monocyte enrichment kit. This study provides evidence to show that the negative selection magnetic separation procedure to isolate monocytes generates the highest yield of monocytes with no significant differences in monocyte viability when compared with monocytes isolated by adherence method. In turn, after seven days, the monocytes isolated by magnetic separation differentiated into MDDCs with significantly higher proliferative capacity and higher amount of cells expressing double positive (CD11c+/CD14+) phenotype without affecting MDDC viability. Overall, the current study differs from the previous studies referenced above since it demonstrates the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into CD11c+ MDDCs (&#62; 70%) after seven days in culture without compromising their viability. In addition, the current approach provides for the first time characterization of different CD11c/CD14 MDDCs populations by imaging flow cytometry.
In summary, since DCs play a focal role regarding research in the field of immunology, different parameters must be taken into consideration when considering how they are derived and what methods are used to isolate and culture them in vitro. Therefore, this study aims to provide insights on two different methods of monocyte isolation and how these methods differentially affect monocyte viability and yield eventually affecting dendritic cell viability, proliferation, and phenotype. These findings will contribute greatly to the field of immunology and will provide a detailed protocol of DC isolation, purification, and characterization.

<table><tbody><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td>17-5442-03</td><td>Must be used at RT</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>ACK Lysing buffer</td><td>Quality Biological</td><td>118-156-101</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Life Technologies</td><td>22400-089</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240-062</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>RoboSep buffer</td><td>StemCell</td><td>20104</td><td></td></tr><tr><td>EasySep Human monocyte enrichment kit</td><td>StemCell</td><td>19059</td><td></td></tr><tr><td>TC20 Automated cell counter</td><td>Bio-Rad</td><td>145-0101</td><td></td></tr><tr><td>FITC-Dextran</td><td>Sigma Aldrich</td><td>FD4-100MG</td><td></td></tr><tr><td>Trypan blue stain (0.4%)</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>Synergy 2 multi-mode reader</td><td>Biotek</td><td>7131000</td><td></td></tr><tr><td>XTT Sodium salt bioreagent (XTT)</td><td>Sigma Aldrich</td><td>X4626-100MG</td><td></td></tr><tr><td>Dimethyl sulfoxide bioreagent (DMS)</td><td>Sigma Aldrich</td><td>D8418-500ML</td><td></td></tr><tr><td>Thiazolyl blue tetrazolium bromide (MTT)</td><td>Sigma Aldrich</td><td>m5655-500MG</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Bio-Rad</td><td>161-0302</td><td></td></tr><tr><td>Phenazine Methosulfate (DMSO)</td><td>Sigma Aldrich</td><td>P9626-1G</td><td></td></tr><tr><td>Inactivated (HI) Human Serum</td><td>Chemicon</td><td>S1-100ML</td><td></td></tr><tr><td>Accuri C6 Flow Cytometer</td><td>BD Accuri</td><td>653119</td><td></td></tr><tr><td>FlowSight Amnis Flow Cytometer</td><td>EMD Millipore</td><td>100300</td><td></td></tr></tbody></table>

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.

Monocyte Yield by Magnetic Separation is Higher Compared to Monocyte Yield by Adherence Method
Data presented in Figure 1 display PBMC and monocyte cell counts by the trypan blue exclusion method at the day of isolation of PBMCs and separation of monocytes. On average, monocytes isolated by the adherence method accounted for approximately 6.2 percent of total PBMCs while monocytes isolated by magnetic separation accounted for up to 25 percent of PBMCs. Statistical analysis revealed that the percentage of monocytes yielded by magnetic separation were significantly higher (&#8805; 4 fold). Data are expressed as mean &#177; SD of at least three independent experiments. Statistical analysis using student&#39;s t-test showed a significant difference of monocyte yield when comparing both methods (p &#8804; 0.0005).
Monocyte Isolation by Adherence and by Magnetic Separation Do Not Affect Monocyte Viability
After monocyte isolation by the two different techniques described above, viability assays were performed to ensure that the methods used were not cytotoxic and affecting the viability of the cells. The data presented in Figure 2 display the average of the percent of viable monocytes isolated by either adherence method or by magnetic separation. Viable cells were counted using the trypan blue exclusion method at the day of isolation. Comparing percent viability of monocytes between the two isolation methods revealed that the difference between both groups was not statistically significant. Additionally, the average of the percent of viable monocytes isolated by adherence was 91.75% &#177; 1.66 and the average of the percent of viable monocytes isolated by magnetic separation was 87.4% &#177; 2.75. These viability data were assessed from at least three independent experiments. Statistical significance was determined using student&#39;s t-test.
Cells Derived from Monocytes Isolated by Magnetic Separation Show a Significant Increase in Cell Proliferation Compared to Cells Derived from Monocytes Acquired by Adherence Method 
MTT was used as a colorimetric assay to measure cell proliferation. In living cells, the yellow tetrazole color of MTT is reduced to purple formazan, which is easily measured using a spectrophotometer. The data presented in Figure 3 demonstrate that both processes of monocyte isolation lead to higher cell proliferation over a period of 7 days as measured by an increased in absorbance at day 0 (adherence method: 0.22 &#177; 0.02 vs. magnetic method: 0.38 &#177; 0.02), day 3 (adherence method: 0.28 &#177; 0.02, magnetic method: 0.55 &#177; 0.05) and day 7 (adherence method: 0.36 &#177; 0.01, magnetic = 0.66 &#177; 0.006). However, unlike the XTT assay (data not shown), the MTT assay revealed a significant increase in cell proliferation of MDDCs from monocytes isolated by magnetic separation compared to MDDCs from monocytes acquired by adherence method at day 0 (p= 0.003), day 3 (p= 0.006), and day 7 (p= 0.002). Cell proliferation was also found to be significantly different between days 0 and 3 (p= 0.003), and between days 0 and 7 (p&#60; 0.001) within the group of MDDCs from monocytes purified through magnetic separation. Furthermore, a significant difference in cell proliferation between day 0 and day 7 in MDDCs from monocytes purified by adhesion (p= 0.008) was observed. MTT assay was conducted using MDDCs acquired from three different experiments. Statistical significance was determined using ANOVA and student&#39;s t-test, p &#8804; 0.05 was considered significant, and p values are noted on the graph unless no statistical significance was found.
Characterization of MDDCs by CD11c and CD14 Cell Surface Staining
After culturing monocytes for seven days with IL-4 and GM-CSF, MDDCs obtained from monocytes isolated by adherence and magnetic isolation methods were characterized in Figure 4. The characterization revealed low single CD14+/CD11c- phenotype or purely monocytic population in both methods, adherence = 1% &#177; 1.0 versus magnetic = 1% &#177; 0.0 and high total CD11c+ phenotype , adherence = 72% &#177; 11 versus magnetic = 79% &#177; 19. However, when the total CD14+ population was analyzed further, there was a high yield of cells double positive for CD11c and CD14 in both methods with magnetic separation giving a higher number of CD11c and CD14 double positive population when compared to the adherence method, adherence = 49% &#177; 7 versus magnetic = 73% &#177; 15. Although, there was a higher yield of MDDCs with double positive phenotype via magnetic separation, the CD11c positive and CD14 negative population of MDDCs was higher in the adherence method; adherence = 23% &#177; 7 versus magnetic = 6% &#177; 7. In Figure 4B, the mean fluorescence intensity (MFI) of each individual marker were looked at in the gated population and showed high intensity of CD14 in CD14+ population, equivalent intensity of both CD11c and CD14 in double positive population and high CD11c intensity in CD11c+ and CD14- population. In summary, even though magnetic isolation gives a higher percentage of CD11c+/CD14+ cells, which may be early stage differentiated MDDCs that have not yet dropped the monocytic marker (CD14), the adherence method gives a higher percentage of CD11c+/CD14-, which may be a late stage differentiated MDDC population. In addition, DAPI staining was performed to confirm viability results and to ensure characterization was performed on live MDDCs. The percent of DAPI negative/live cells (adherence = 76 &#177; 7 versus magnetic = 67 &#177; 1) are not significantly different between the two methods confirming both methods do not affect the viability of the MDDCs after seven days in culture.
<img alt="Figure 1" src="/files/ftp_upload/54296/54296fig1.jpg" /> 
Figure 1. Monocyte Yield by Magnetic Separation is Higher Compared to Monocyte Yield by Adherence Method. About 6.2% of monocytes were isolated from PBMCs using the adherence method while ~ 25% of monocytes were obtained using the magnetic separation method. Data are expressed as mean &#177; SD of at least three independent experiments. Statistical analysis using student&#39;s t-test showed a significant difference of monocyte yield when comparing both methods (p = 0. 00005). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54296/54296fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54296/54296fig2.jpg" /> 
Figure 2. Monocyte Isolation by Adherence and by Magnetic Separation Do Not Affect Monocyte Viability. The average of the percent of viable monocytes isolated by adherence was 91.75% &#177; 1.66 and the average of the percent of viable monocytes isolated by magnetic separation was 87.4% &#177; 2.75. Data are expressed as mean &#177; SD percentage of viable cells of at least three independent experiments. No significant difference was observed in cell viability when comparing both purification methods. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54296/54296fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54296/54296fig3.jpg" /> 
Figure 3. Cells Derived from Monocytes Isolated by Magnetic Separation Show a Significant Increase in Cell Proliferation Compared to Cells Derived from Monocytes Acquired by Adherence Method. A significant increase in absorbance was observed for both methods at both day 3 and day 7 when comparing the values against its respective controls of day 0. In addition, ANOVA and student&#39;s t-test also showed a significant difference in absorbance of MTT uptake when comparing both methods (p &#8804; 0.05). Data are expressed as mean &#177; SD of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54296/54296fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54296/54296fig4.jpg" /> 
Figure 4. Characterization of MDDCs by CD11c and CD14 Cell Surface Staining. A) Bar graph representing the different populations (% gated out of single cells). First, DAPI-(alive) cells were gated from total single cells. Then, CD14+/CD11c-, CD11c+, CD11c+/CD14+ and CD11c+/CD14- were gated from DAPI- (alive) population. CD11c + population is the summation of two regions CD11c+/CD14+ and CD11c+/CD14-. B) Representative graphs showing the mean fluorescence intensity (MFI) values for both CD11c and CD14 in every subpopulation of cells. C) Representative scatter plots of cells labelled with CD11c-APC and CD14-APCcy7 gated on DAPI negative (alive) cell population for both magnetic and adherence methods. Representative image gallery of single cells (selected in blue in the scatter plots) belonging to each sub-population of cells, CD11c+/CD14- (gated in orange), CD11c+/CD14+ (gated in red), and CD14+ (gated in purple). In the image gallery of single cells, the red color represents CD11c labelled with APC and yellow color represents CD14 labelled with APCcy7. In the scatter plots, the colors chosen to gate the populations are random and do not correlate to the actual cell images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54296/54296fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols at JoVE.com

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

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

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

characterization-human-monocyte-derived-dendritic-cells-imaging-flow

Nanyang Technological University

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JoVE Journal

Immunology and Infection

13.5K Views.  Florida International University. 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.

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

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

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.

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 ...

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

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Bioengineering

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

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 ...

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

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