Name: simultaneous study of the recruitment of monocyte subpopulations under flow in vitro
Uploaded: 2018-11-26T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro at JoVE.com

We thank Dr. Paul Bradfield for manuscript reading and feedbacks. A. S. received financial support from the Sir Jules Thorn Charitable Overseas Trust Reg.,

Human materials were used with the informed consent of volunteer donors and in accordance with the Swiss Ethics Committees on clinical research.
1. Isolation and Freezing of Human Umbilical Vein Endothelial Cells (HUVEC)
<ol>
	<li>Add 5 mL of coating solution to a T75 flask (0.1 mg/mL collagen G and 0.2% gelatin in phosphate buffered saline PBS at pH 7.4) for 30 min at 37 &#176;C before initiating HUVEC isolation.</li>
	<li>Clean the cord with PBS, wipe it with sterile compresses, and place ...

<ol>
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Here, we report a method detailing a study of how monocyte subpopulations transmigrate through the inflamed endothelial monolayer. The discussed method used confocal microscopy instead of phase-contrast microscopy, which is also used to study monocyte recruitment under flow3,11,19. One major advantage of using confocal microscopy for time-lapse imaging is the ability to unambiguously discriminate between transmigration and stron...

Monocytes constitute a phagocytic component of innate immunity that is essential for fighting pathogens, cleaning up damaged tissues, angiogenesis, and the pathophysiology of many diseases including cancers1,2,3. Monocytes are bone marrow-derived cells composed of heterogeneous subpopulations that circulate in the blood but can be recruited to the site of inflammation in peripheral tissue through specific molecular mechanisms. The recruitment cascades of monocytes, as for leukocytes in general, implicates different steps including capture, rolling, crawling, arrest, transendothelial migration (transmigration) and migration through the vessel wall (basement membrane and mural cells)4. These steps mainly involve inflammation-induced molecules on the endothelial luminal surface such as selectins, glycoprotein ligands, chemokines, intercellular and junctional adhesion molecules, and their receptors on leukocytes such as selectin ligands and integrins. Trafficking pathways through either the endothelial cell junctions (paracellular) or through the endothelial cell body (transcellular) can be used by leukocytes to cross the endothelial barrier5. Whilst monocytes have historically been documented to transmigrate through the transcellular route, potential divergences in their migratory pathway have been proposed as monocytes are no longer considered a homogeneous cell population. It is now becoming clear that monocyte diversity can be defined by each of their differences and commonalities, with respect to their distinctive extravasation cascades3,6. Therefore, in order to unambiguously discriminate between monocyte subpopulations, it is crucial to visualize and phenotype the behavior of each of these different subpopulations during the recruitment process.
Monocytes from human, pig, rat and mouse were subdivided into phenotypic subpopulations with certain ascribed functions and specific migratory behaviors7,8,9. For example, in humans, monocytes can be divided into three subsets based on their surface expression of CD14, a coreceptor for bacterial lipopolysaccharide, and CD16, the Fc-gamma receptor III. Human monocyte subpopulations include classical CD14+CD16-, intermediate CD14+CD16+ and non-classical CD14dimCD16+ cells6,9. The classical CD14+CD16- monocytes were shown to be mainly inflammatory whereas the pool of CD16+ monocytes were collectively found to present TIE2 expression and proangiogenic function10. Consistently, endothelial cell stimulation with inflammatory cytokines such as human tumor necrosis factor (TNF)&#945; or interleukin (IL-1)beta (conventional inflammation) is sufficient to trigger the complete recruitment of classical CD14+CD16- monocytes. However, simultaneous actions of vascular endothelial growth factor (VEGF)A and TNF&#945; (angiogenic factors-driven inflammation) are required to provoke the transmigration of the CD16+ proangiogenic pool of monocytes3. Historically, the traditional Transwell system under static conditions, the parallel plate flow chamber, and the &#181;-slide flow chambers have been used to quantitatively analyze the recruitment of one leukocyte population at a time in vitro11,12,13. Whilst these protocols have been validated, a more robust method that allowed the simultaneous analysis of multiple monocyte subpopulations would be considered more insightful. Such methodologies must account for multiple interactions and the differing frequencies of each respective population and also provide a mechanistic understanding of the similarities and specificities for the recruitment cascades that define each monocyte subset.
Here, we present a method based on the time-lapse imaging of monocyte recruitment under flow which allows the migratory cascades of different monocyte subpopulations to be studied simultaneously by using confocal microscopy. This method integrates certain critical features that mimic endothelial cell inflammation, as well as the hemodynamics of circulating monocytes in post-capillary venules, the main location of leukocyte recruitment in vivo. The proposed method uses human umbilical vein endothelial cells (HUVEC), which are generated through a well-established protocol of isolation from human umbilical cords. This clinical resource has the advantage of being easily available as a biological by-product, whilst also providing a reasonable yield of endothelial cells that can be isolated from the umbilical vein. We also used fluorescent dyes and immunofluorescence to distinguish between the different cellular components, and confocal microscopy to unambiguously define monocyte positioning (luminal vs. abluminal) over time. The protocol presented here has been developed to simultaneously measure the transmigration levels of monocyte subpopulations. Moreover, it should be noted that this methodology can be extended to study other leukocytes subpopulations and recruitment processes by use of different biomarkers and labelling.

<table>
	<tbody>
		<tr>
			<td>Tissue Culture Flasks 75 cm2</td>
			<td>TPP</td>
			<td>90076</td>
			<td>Routine culture of isolated HUVEC</td>
		</tr>
		<tr>
			<td>&#181;-Slide VI 0.4</td>
			<td>IBIDI</td>
			<td>80606</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes 15 mL</td>
			<td>TPP</td>
			<td>191015</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes 50 mL</td>
			<td>TPP</td>
			<td>191050</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen G</td>
			<td>Biochrom</td>
			<td>L 7213</td>
			<td>For coating of cell culture flasks</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>1393</td>
			<td>For coating of cell culture flasks</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (without MgCl2 and CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (with MgCl2 and CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Way Stopcocks</td>
			<td>BIO-RAD</td>
			<td>7328103</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin 10000 u/ml streptomycine 10000 ug/ml fungizone 25 ug/ml</td>
			<td>AMIMED</td>
			<td>4-02F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type 1</td>
			<td>Worthington</td>
			<td>LS004216</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199 1X avec Earle&#39;s salts, L-Glutamine, 25 mM Hepes</td>
			<td>GIBCO</td>
			<td>22340020</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Albumin Fraction V</td>
			<td>ThermoFisher</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Supplement, 150mg</td>
			<td>Millipore</td>
			<td>02-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium</td>
			<td>Sigma-Aldrich</td>
			<td>H3149RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H6909</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A 4544</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate C10H14N2Na2O8 &#183; 2H2O</td>
			<td>APPLICHEM</td>
			<td>A2937.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>CD144 (VE-Cadherin), human recombinant clone: REA199, FITC</td>
			<td>Miltenyi Biotech</td>
			<td>130-100-713</td>
			<td>AB_2655150</td>
		</tr>
		<tr>
			<td>CD31-PE antibody, human recombinant clone: REA730, PE</td>
			<td>Miltenyi Biotech</td>
			<td>130-110-807</td>
			<td>AB_2657280</td>
		</tr>
		<tr>
			<td>Anti-Podoplanin-APC, human recombinantclone: REA446, APC</td>
			<td>Miltenyi Biotech</td>
			<td>130-107-016</td>
			<td>AB_2653263</td>
		</tr>
		<tr>
			<td>BD Accuri C6 Plus</td>
			<td>BD Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide I Luer</td>
			<td>IBIDI</td>
			<td>80176</td>
			<td></td>
		</tr>
		<tr>
			<td>CMFDA (5-chloromethylfluorescein diacetate)</td>
			<td>ThermoFisher</td>
			<td>C2925</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human TNF&#945;</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human VEGFA</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>NE-1000 Programmable Syringe Pump</td>
			<td>KF Technology</td>
			<td>NE-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll&#160;Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD14-PE, human recombinant clone: REA599, PE</td>
			<td>Miltenyi Biotech</td>
			<td>130-110-519</td>
			<td>AB_2655051</td>
		</tr>
		<tr>
			<td>Pan Monocyte Isolation Kit, human</td>
			<td>Miltenyi Biotech</td>
			<td>130-096-537</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD16-PE, human recombinant clone: REA423, PE</td>
			<td>Miltenyi Biotech</td>
			<td>130-106-762</td>
			<td>AB_2655403</td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotech</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>ThermoFisher</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tubing</td>
			<td>IBIDI</td>
			<td>10841</td>
			<td></td>
		</tr>
		<tr>
			<td>Elbow Luer Connector</td>
			<td>IBIDI</td>
			<td>10802</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Luer Lock Coupler</td>
			<td>IBIDI</td>
			<td>10823</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock Connector Female</td>
			<td>IBIDI</td>
			<td>10825</td>
			<td></td>
		</tr>
		<tr>
			<td>In-line Luer Injection Port</td>
			<td>IBIDI</td>
			<td>10820</td>
			<td></td>
		</tr>
		<tr>
			<td>Ar1 confocal microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40X objective</td>
			<td>Nikon</td>
			<td>40x 0.6 CFI ELWD S Plane Fluor WD:3.6-2.8mm correction 0-2mm</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
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simultaneous study of the recruitment of monocyte subpopulations under flow in vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Determining the state of HUVEC activation induced by TNF&#945;
The bio-activity of the inflammatory cytokine TNF&#945; can be vary according to the batch and the repletion of freezing-thawing cycle. It is important to check the activation status of HUVEC with TNF&#945; treatment. This could be performed by staining in parallel some samples of confluent HUVEC for the inflammatory induction of selectins, ICAM-1 and VCAM-1<sup class="...

Watch this Scientific Journal Video about Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro at JoVE.com

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

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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 ...

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 ...