Name: isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags
Uploaded: 2014-09-09T13:00:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags at JoVE.com

The authors would like to thank Mrs. Meike Schaffrinski for her always excellent technical assistance during the last few years.
This work was funded through the German Research Council (DFG) within the joint research group 942 (FOR942) and by the Research Program of the Faculty of Medicine, Georg-August-University G&#246;ttingen.

1. Preparation of Sterile Human AB Serum
<ol>
	<li>Collect 4-5 bags of fresh frozen plasma (FFP). Store bags at -20&#160;&#176;C until enough bags have been collected.</li>
	<li>Thaw bags and incubate for 30&#160;min at 56&#160;&#176;C in a water bath to inactivate complement and remove fibrin.</li>
	<li>Thoroughly disinfect the outside of the bags and transfer the plasma to 50&#160;ml tubes.</li>
	<li>Centrifuge at 3,000&#160;x&#160;g for 15&#160;min at room temperature to get rid of precipitates and residual platelets.</li>
	<li>Pool all supernatants and discard the pellets.</li>
	<li>Aliquot serum samples in 15&#160;ml tubes and store at -20&#160;&#176;C. 
	NOTE: Alternatively, commercially available heat-inactivated normal human AB serum can be used.</li>
</ol>
2. Isolation of Monocytes
To facilitate balancing of the centrifuge, it is recommended to process two buffy coats in parallel. However, take care to use separate materials for each donor and not to mix the cells. In case buffy coats cannot be obtained easily, 400&#160;ml of heparinized peripheral blood can be used instead.
<ol>
	<li>Carefully disinfect the plastic bags containing the buffy coats and transfer the contents of each buffy coat to two 50&#160;ml tubes.</li>
	<li>For each buffy coat fill three 50&#160;ml tubes with 15&#160;ml Ficoll solution (1.077&#160;g/ml). The Ficoll should be at room temperature for the preparation.</li>
	<li>Layer 30-35&#160;ml of the buffy coat blood on top of the Ficoll solution for the first density gradient. Be careful to do this slowly and carefully in order to prevent mixing both layers.</li>
	<li>Centrifuge at 400&#160;x&#160;g without brake for 30&#160;min at room temperature.</li>
	<li>For each gradient collect the white ring of peripheral blood mononuclear cells (PBMCs) which is located between the two phases with a plastic Pasteur pipette and transfer to a 50&#160;ml tube.</li>
	<li>Fill each tube with PBS-EDTA (1 mM) up to 40&#160;ml in total.</li>
	<li>Centrifuge at 300&#160;x&#160;g for 10&#160;min without brake at room temperature.</li>
	<li>Aspirate supernatant and wash pellet again with 40&#160;ml PBS-EDTA (1 mM).</li>
	<li>For each donor pool the pellets in 20&#160;ml RPMI-1640 without phenol red +&#160;10% FCS.</li>
	<li>Prepare the iso-osmotic Percoll solution for the second density gradient: For two donors mix 23.13&#160;ml Percoll solution (density: 1.131 g/ml) in a 50&#160;ml tube with 1.87&#160;ml 10-fold PBS. Then transfer 23&#160;ml of this solution to a new 50&#160;ml tube and add 27&#160;ml RPMI-1640 with phenol red + 10% FCS to obtain a 46% iso-osmotic Percoll solution. The Percoll should be at room temperature for the preparation.</li>
	<li>For each donor transfer 25&#160;ml of the prepared Percoll solution to a 50&#160;ml tube and layer the PBMC solution prepared in 2.9) on top of the Percoll solution. Be careful to do this very slowly and carefully, both layers tend to mix easily. If done correctly the two phases can be distinguished due to their difference in color.</li>
	<li>Centrifuge at 550&#160;x&#160;g without brake for 30&#160;min at room temperature.</li>
	<li>For each gradient collect the white ring of monocytes which is located between the two phases with a plastic Pasteur pipette and transfer to a 50&#160;ml tube.</li>
	<li>Fill each tube with PBS-EDTA (1&#160;mM) up to 50&#160;ml in total.</li>
	<li>Centrifuge at 400&#160;x&#160;g for 10&#160;min without brake at room temperature.</li>
	<li>Aspirate the supernatant and resuspend the pellets in 20&#160;ml RPMI-1640 with phenol red + 10% FCS.</li>
</ol>
3. Differentiation of Monocytes to Macrophages
<ol>
	<li>Determine the number of isolated monocytes in a 1:10 dilution in trypan blue. Only count the large, often irregular-shaped cells, which are monocytes. Do not count the smaller, round-shaped cells which are lymphocytes. 
	NOTE: Monocyte numbers do not have to be determined too accurately because they only give a hint about how many and which FEP Teflon-coated bags (small or large) have to be used for the differentiation of the cells.</li>
	<li>For 1.0-1.5 x&#160;108 monocytes from one donor, seed the cells in a large FEP Teflon-coated cell culture bag. For each bag prepare the culture medium consisting of 174&#160;ml RPMI-1640, 2% human AB serum (as prepared in Section 1), 1% penicillin/streptomycin, and 2.5&#160;ng/ml M-CSF (total volume: 180&#160;ml).
	<ol>
		<li>For 3.0-5.0 x&#160;107 monocytes from one donor, seed the cells in a small Teflon-coated cell culture bag. For each bag prepare the culture medium consisting of 28.5&#160;ml RPMI-1640, 2% human AB serum (as prepared in Section 1), 1% penicillin/streptomycin, and 2.5&#160;ng/ml M-CSF (total volume: 30&#160;ml). 
		NOTE: If e.g., 8.0 x&#160;107 monocytes are obtained, we recommend to seed them in two smaller volume bags with 4.0 x&#160;107 cells each. Instead of M-CSF, monocytes can alternatively be differentiated with granulocyte macrophage colony-stimulating factor (GM-CSF) at the same concentration (2.5&#160;ng/ml).</li>
	</ol>
	</li>
	<li>Add the cell suspension (20 ml) to the prepared medium and mix carefully. If two bags are prepared from one donor, add 20&#160;ml RPMI-1640 to the cell suspension and then add half of the suspension to each medium preparation.</li>
	<li>Optional: To determine if the monocyte preparation has remained sterile, tear one drop of the cell suspension on a blood agar plate and incubate over night at 37&#160;&#176;C.</li>
	<li>Tighten the sheath of a 50&#160;ml perfusor syringe onto the plug of the FEP Teflon-coated cell culture bag and fill it with the prepared cell suspension.</li>
	<li>Unplug the syringe and push the remaining air out of the bag. Close the bag with a closing cone.</li>
	<li>Incubate the bags for 6-7 days at 37&#160;&#176;C with 5% CO2.</li>
</ol>
4. Macrophage Harvest
<ol>
	<li>Place the FEP Teflon-coated cell culture bags with the differentiated macrophages on ice for at least 1&#160;hr to facilitate detachment of the cells (an incubation of up to 3&#160;hr is possible). Make sure that the whole surface of the bag is covered with ice.</li>
	<li>Pull the bag with minimal pressure 10x&#160;over the edge of a desk/board.</li>
	<li>Carefully disinfect the outside of the bag and remove the closing cone.</li>
	<li>Tighten a 50&#160;ml syringe on the plug of the bag, remove the cell suspension, and transfer it to 50&#160;ml tubes.</li>
	<li>Centrifuge at 400&#160;x&#160;g for 10&#160;min at room temperature to spin down the cells.</li>
	<li>Aspirate the supernatant and pool the pellets from one bag in 10&#160;ml RPMI-1640 + 10%&#160;FCS in total.</li>
	<li>Determine the cell number in a hemocytometer (1:4-1:10 dilution in trypan blue). Only count the large round cells. 
	NOTE: After differentiating the macrophages in the FEP Teflon-coated bags, there can be residual cells e.g., lymphocytes, depending on the donor and on the quality of the monocyte isolation.</li>
	<li>To reuse the FEP Teflon-coated bags, wash them twice with 70% ethanol to remove residual cells. Fill them with 50&#160;ml 70% ethanol and incubate overnight. Wash the bags 3x with sterile PBS, wrap them in sterilization paper, and sterilize in an autoclave using standard procedures. 
	NOTE: The cell culture bags can be reused several times without any loss in macrophage yields. However, we decided to discard the bags after 10 uses before they start to&#160;leak.</li>
	<li>For subsequent experiments culture macrophages in RPMI-1640 + 10% FCS. If lower serum concentrations are required, cultivation in RPMI-1640 with 1% FCS is also possible. Seed and incubate cells for 3-4 hr. After this period, note that macrophages attach to the surface of the cell culture dish while any contaminating cells (i.e. erythrocytes, lymphocytes, and dendritic cells) stay in suspension. Wash cells at least twice with PBS to remove non-adherent cells.</li>
	<li>To detach cultured macrophages from the cell culture dish for further experiments, carefully scrape them off the surface. Alternatively, incubate dishes for 20&#160;min on ice, aspirate the culture medium, and add 1&#160;ml enzyme-free cell dissociation buffer. After 5&#160;min incubation at 37 &#176;C, add 4&#160;ml PBS, and detach cells by pipetting up and down for at least 3&#160;min.</li>
</ol>

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The authors have nothing to disclose as no conflict of interests exists.

Macrophages are important effector cells of the innate immune system and display important functions in immunomodulation, antigen presentation and tissue homeostasis. Due to their remarkable plasticity, they are able to respond to different stimuli with changes of their phenotype. However, so far a lot of data regarding macrophage polarization are obtained in the murine system, although there are reports showing that only around 50% of macrophage polarization markers can be directly translated from mouse to human16. Therefore, we present here a method to obtain primary human macrophages in sufficient number and purity without the need for expensive materials,&#160;e.g., MACS magnetic beads or a counterflow centrifugal elutriation device.
Our method is based on the isolation of monocytes from PBMCs and their subsequent differentiation to macrophages in FEP Teflon-coated cell culture bags in the presence of low concentrations of M-CSF11-13. While monocytes make up less than 5 to 10% of peripheral blood leukocytes in humans, upon stimulation they are recruited to peripheral sites where they differentiate to resident tissue macrophages or dendritic cells17. The cytokine M-CSF is important for monocyte survival and drives their differentiation to macrophages18,19. So far, the M-CSF concentrations which were chosen for monocyte differentiation ranged up to 100&#160;ng/ml, however, in our protocol we are able to obtain sufficient numbers of mature macrophages with a M-CSF concentration of only 2.5&#160;ng/ml12,20. In addition, cells are cultured in FEP Teflon-coated cell culture bags which facilitate the detachment of the macrophages and their subsequent seeding in defined cell numbers. Since the bags can be reused several times, this further decreases the costs for the isolation process.
The macrophages obtained by this procedure are highly positive for CD45, CD14, CD11b, CD11c and show expression of the mannose receptor CD206 which argues for a population of pure, mature macrophages21,22. Especially high CD14 expression is typical for macrophages differentiated in the presence of M-CSF23. After seeding of the cells, they display a fast adherence to plastic surfaces with some cells showing a typical spindle-like morphology, while others exhibit a fried egg phenotype. This is in accordance with the observations from other authors18,22,24.
It was reported that monocyte differentiation in the presence of M-CSF leads to M2-polarized macrophages16,25. However, the macrophages isolated by our protocol are still able to respond to a wide range of stimuli including exposure to tumor cell-derived microvesicles and co-culture with tumor cells26,27 or exposure to LPS to which they react with induction of pro-inflammatory genes such as IL-1&#946;, TNF&#945;, Wnt5a, or various matrix metalloproteinases which are considered typical for M1-polarized macrophages3,5,28.
In conclusion, the isolation of monocytes by double density gradient centrifugation and subsequent differentiation towards macrophages in FEP Teflon-coated cell culture bags results in high numbers of macrophages without the need for technically difficult or expensive procedures. The obtained macrophages can be utilized for subsequent analysis ranging from classical activation through LPS to the co-culture with tumor cells.

Cells from the monocytic lineage and their terminally differentiated derivative -&#160;macrophages&#160;- exhibit a striking plasticity in regard to their biological function, leading to their involvement in such diverse processes as development, tissue repair, and immunity1. The latter is due to their phagocytic and antigen-presenting ability which places macrophages at the crossroads between the innate and adaptive immune response2. However, their capability to secrete cytokines, chemokines, growth factors, and other signaling molecules1 not only augments their immune-modulatory function but also serves as a basis for their additional functions. Attempts to mirror these diverse activation steps in the context of non-microbial mediated conditions have resulted in the M1 and M2 categories3. While this classification is not complete, it allows for a basic understanding of macrophage biology.
Due to these multifaceted capabilities it comes as no surprise that macrophages are associated with many conditions that in some way involve tissue remodeling or inflammation. Next to their fundamental role in the recognition and clearance of invading pathogens4-6, macrophages have increasingly come into focus in atherosclerosis, fibrosis, obesity, and cancer7-10. A reproducible method for the generation of human macrophages is therefore crucial for an understanding of these pathologies. Here we present a method based on the isolation of human monocytes from the peripheral blood of healthy donors by a double density gradient centrifugation technique as described previously11. In order to facilitate differentiation towards macrophages, the isolated monocytic cells are incubated in the presence of low concentrations of M-CSF and normal human serum12. To ease further handling and cell harvest, differentiation is carried out in gas permeable FEP Teflon-coated cell culture bags with a hydrophobic surface12-15. The resulting resting macrophages can be subjected to a wide range of assays as they are still capable of responding in either an M1 or M2-like fashion. Alternative methods of monocyte isolation and subsequent differentiation such as magnetic activated cell sorting (MACS) or counterflow centrifugal elutriation (CCE) have some limitations regarding the yield, cost, and time required. The protocol described herein offers the advantage that it can be carried out with standard laboratory equipment without the need for special reagents (e.g., MACS magnetic beads) or devices (e.g., CCE apparatus) and allows for the processing of large quantities of cells.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>antibodies for immunophenotyping</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>for example: CD11c-PE (IM1760), CD45-FITC (7782), IgG1-PE isotype control (A07796), IgG1-FITC isotype control (A07795)</td>
		</tr>
		<tr>
			<td></td>
			<td>BioLegends</td>
			<td></td>
			<td>for example: CD14-FITC (325603), CD206-PE (321105)</td>
		</tr>
		<tr>
			<td>Axiovert 200M microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>calcein-AM</td>
			<td>AnaSpec</td>
			<td>89201</td>
			<td></td>
		</tr>
		<tr>
			<td>combi-stopper closing cones</td>
			<td>Braun</td>
			<td>4495101</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS, w/o Ca and Mg</td>
			<td>Pan biotech</td>
			<td>P04-36500</td>
			<td>for PBS-EDTA (1 mM) add 1 ml 0,5 M EDTA per 500ml PBS</td>
		</tr>
		<tr>
			<td>10x PBS, w/o Ca and Mg</td>
			<td>Invitrogen</td>
			<td>14200-067</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (Titriplex III)</td>
			<td>Merck</td>
			<td>1084211000</td>
			<td>prepare a 0,5 M solution in H2O, use a sterile filter</td>
		</tr>
		<tr>
			<td>cell dissociation buffer (enzyme-free, PBS-based)</td>
			<td>Gibco</td>
			<td>13151-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Invitrogen</td>
			<td>10091148</td>
			<td>heat-inactivated</td>
		</tr>
		<tr>
			<td>Ficoll (density 1.077g/ml)</td>
			<td>Biochrom AG</td>
			<td>L6115</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCanto II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse FITC</td>
			<td>santa cruz</td>
			<td>sc-2010</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from E.coli</td>
			<td>Sigma</td>
			<td>L8274</td>
			<td>final conc: 100 ng/ml</td>
		</tr>
		<tr>
			<td>Multifuge 3 L-R</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Biochrom AG</td>
			<td>A2213</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (density 1,131g/ml)</td>
			<td>GE Healthcare</td>
			<td>17-0891-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusor syringe 50ml</td>
			<td>Braun</td>
			<td>8728844F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-TRITC</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>resuspend in methanol (c = 0,1 mg/ml)</td>
		</tr>
		<tr>
			<td>Plastic Disposable Pasteur Pipettes</td>
			<td>LVL technologies</td>
			<td>2655181</td>
			<td></td>
		</tr>
		<tr>
			<td>rh M-CSF</td>
			<td>ImmunoTools</td>
			<td>11343117</td>
			<td>Resuspend in 500&#181;l sterile H2O (c = 100 ng/&#181;l), aliquot</td>
		</tr>
		<tr>
			<td>RPMI-1640 with Phenol Red</td>
			<td>Gibco</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 without Phenol Red</td>
			<td>Gibco</td>
			<td>11835-063</td>
			<td></td>
		</tr>
		<tr>
			<td>sterilization paper</td>
			<td>VP group</td>
			<td>3KFKFS230116</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue stain (0,4% w/v)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>FEP Teflon-coated cell culture bag, small</td>
			<td>CellGenix</td>
			<td>72-C</td>
			<td></td>
		</tr>
		<tr>
			<td>FEP Teflon-coated cell culture bag, large</td>
			<td>CellGenix</td>
			<td>197-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The first density gradient centrifugation using Ficoll yields a white interphase containing the PBMCs (Figure&#160;1A) i.e. lymphocytes and monocytes. This can be confirmed through a May-Gruenwald staining (Figures&#160;1B&#160;and&#160;C) of the collected cells which shows both a high nucleus/cytoplasm ratio (typical of lymphocytes) and bean- or ring-shaped nuclei (typical of monocytes). When these cells are then loaded onto a second density gradient using Percoll, the monocytes can be further separated from the lymphocytes and again appear as a white interphase (Figures 1D-F). For each buffy coat the described double density gradient centrifugation routinely yields 150&#160;&#177;&#160;40 x&#160;106 monocytes which can be differentiated to 70&#160;&#177;&#160;30 x&#160;106 macrophages (Figure&#160;2) per buffy coat. The mean macrophage yield from 20 independent preparations was 47 &#177; 14% of total isolated monocytes.
After the Percoll gradient centrifugation there might still be some residual non-monocytic cells present in the preparation which is dependent on the blood donor as well as on the accuracy of the isolation process. However, after the differentiation phase of 6-7 days, the preparation mainly consists of&#160;mature macrophages (Figure&#160;3) which can be further enriched due to their adherence to plastic surfaces, a feature that is not shared by the random contaminating cells (Figures&#160;4A and B). Once plated, the majority of the macrophages show a classical &#8220;fried egg&#8221; morphology while there are also cells with a stretched spindle-like phenotype (Figures&#160;4C and D). This is mirrored by their F-actin distribution within the cytoplasm and adhesion clusters. The differentiated cells are characterized by the expression of CD45, CD14, CD16, CD206 (mannose receptor), CD11b and CD11c which are typical markers for mature macrophages (Figure&#160;5). The presence of CD11b argues against a predominantly dendritic differentiation which is supported by the fact that the cells are negative for the dendritic cell marker CD209 (DC-SIGN).
After the differentiation process the cells remain functionally and metabolically active for approximately 5-7 days (Figure&#160;6) as it can be visualized by calcein&#160;AM staining and their ability to take up extracellular vesicles shed from tumor cells. Additionally, the cells can still be activated as shown e.g., for the stimulation with lipopolysaccharide (LPS) which results in the expression of several pro-inflammatory genes (Figure&#160;7).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51554/51554fig1highres.jpg" width="500" /> 
Figure 1.&#160;Appearance and composition of the PBMC- and monocyte-layer after double density gradient centrifugation. Photograph illustrating (A) the PBMC-band after the Ficoll gradient and (D) the monocyte-phase after the iso-osmotic Percoll centrifugation. May-Gruenwald stainings of cytospin preparations of the (B,&#160;C) PBMC fraction and the remaining (E,&#160;F) monocytes. Scale bar = 200 &#181;m in B and&#160;E, = 50 &#181;m in C and&#160;F.
<img alt="Figure 2" fo:content-width="3in" src="/files/ftp_upload/51554/51554fig2highres.jpg" width="300" /> 
Figure 2.&#160;Yield of monocytes and macrophages. Representative cell countings of isolated monocytes and macrophages of 20 buffy coat preparations.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51554/51554fig3highres.jpg" width="600" /> 
Figure 3.&#160;Micrographs and cell size measurements of monocytes and macrophages. Phase contrast microscopy of monocytic cell suspension before (A) and after (B) macrophage differentiation. Corresponding cell size histograms of monocytes (C) and macrophages (D). Scale bar = 100&#160;&#181;m.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51554/51554fig4highres.jpg" width="600" /> 
Figure 4.&#160;Morphology and cytoskeletal organization of adherent macrophages. Phase contrast microscopy of adherent macrophages before (A) and after (B) removal of non-adherent cells. (C,&#160;D) Phalloidin-TRITC staining of filamentous actin in adherent, non-stimulated macrophages. Scale bar = 100 &#181;m in A-C, 20 &#181;m in D. <a href="https://www.jove.com/files/ftp_upload/51554/51554fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51554/51554fig5highres.jpg" width="500" /> 
Figure 5.&#160;Immunophenotype of differentiated macrophages. Flow cytometry analysis of macrophages after 6 days of differentiation in FEP Teflon-coated cell culture bags (indicated in red). The corresponding isotype controls are shown in grey.&#160;<a href="https://www.jove.com/files/ftp_upload/51554/51554fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" fo:content-width="6in" src="/files/ftp_upload/51554/51554fig6highres.jpg" width="600" /> 
Figure 6.&#160;Uptake of tumor cell microvesicles by macrophages. Micrographs of adherent macrophages after exposition to PKH26-labeled (red fluorescent) tumor cell-derived microvesicles. Images are overlayed to the corresponding (A) brightfield or (B) cytosolic staining with the viability dye calcein&#160;AM. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/51554/51554fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51554/51554fig7highres.jpg" width="500" /> 
Figure 7.&#160;Upregulation of IL-1&#946;, Wnt5a, TNF&#945;, IL-6, MMP-2, MMP-7, and MT1-MMP after stimulation of macrophages with LPS (100 ng/ml) for 24 hr. Gene expression was measured by quantitative RT-PCR from total RNA samples (A) and normalized on HPRT1 and GNB2L1 expression. The values shown are fold changes in comparison to the untreated control (means &#177; SD, n=5, *p&#60;0.05, **p&#60;0.01, ***p&#60;0.001). TNF&#945; and IL-6 induction under LPS stimulation were further confirmed by ELISA (B) (means &#177; SD, *p&#60;0.05).

Watch this Scientific Journal Video about Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags at JoVE.com

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

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 ...

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Research

JoVE Journal

Immunology and Infection

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

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 ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

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 ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

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. ...