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 disadvan...

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.

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			<td height="34" style="height:34px;">Phosphate-buffered saline (PBS)</td>
			<td>Life Technologies</td>
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			<td></td>
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			<td height="34" style="height:34px;">ACK Lysing buffer</td>
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			<td></td>
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			<td height="34" style="height:34px;">RPMI 1640 medium</td>
			<td>Life Technologies</td>
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			<td height="34" style="height:34px;">Antibiotic-Antimycotic (100X)</td>
			<td>Life Technologies</td>
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			<td></td>
		</tr>
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			<td height="34" style="height:34px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td></td>
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			<td height="34" style="height:34px;">RoboSep buffer</td>
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			<td>20104</td>
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			<td height="34" style="height:34px;">EasySep Human monocyte enrichment kit</td>
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			<td height="34" style="height:34px;">TC20 Automated cell counter</td>
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			<td height="34" style="height:34px;">FITC-Dextran</td>
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			<td>FD4-100MG</td>
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			<td height="34" style="height:34px;">Trypan blue stain (0.4%)</td>
			<td>Life Technologies</td>
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			<td height="34" style="height:34px;">Synergy 2 multi-mode reader</td>
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			<td>Sigma Aldrich</td>
			<td>X4626-100MG</td>
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			<td height="34" style="height:34px;">Dimethyl sulfoxide bioreagent (DMS)</td>
			<td>Sigma Aldrich</td>
			<td>D8418-500ML</td>
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			<td height="34" style="height:34px;">Thiazolyl blue tetrazolium bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>m5655-500MG</td>
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			<td height="34" style="height:34px;">Sodium Dodecyl Sulfate (SDS)</td>
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			<td>161-0302</td>
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			<td>Chemicon</td>
			<td>S1-100ML</td>
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		<tr height="34">
			<td height="34" style="height:34px;">Accuri C6 Flow Cytometer</td>
			<td>BD Accuri</td>
			<td>653119</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FlowSight Amnis Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>100300</td>
			<td></td>
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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 ma...

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 ...

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Immunology and Infection

13.4K 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

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

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

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

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

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

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

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

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

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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 ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

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

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

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

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

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...