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 ...

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I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nature Review Immunology. 7 (9), 678-689 (2007).</li><li>Nourshargh, S., Alon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+Migration+into+Inflamed+Tissues.">Leukocyte Migration into Inflamed Tissues.</a> Immunity. 41 (5), 694-707 (2014).</li><li>Cros, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD14dim+monocytes+patrol+and+sense+nucleic+acids+and+viruses+via+TLR7+and+TLR8+receptors.">Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors.</a> Immunity. 33 (3), 375-386 (2010).</li><li>Geissmann, F., Jung, S., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+monocytes+consist+of+two+principal+subsets+with+distinct+migratory+properties.">Blood monocytes consist of two principal subsets with distinct migratory properties.</a> Immunity. 19 (1), 71-82 (2003).</li><li>Chamorro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+porcine+blood+CD163&#8722;+and+CD163++monocytes+into+functional+dendritic+cells.">In vitro differentiation of porcine blood CD163&#8722; and CD163+ monocytes into functional dendritic cells.</a> Immunobiology. 209 (1-2), 57-65 (2004).</li><li>Passlick, B., Flieger, D., Ziegler-Heitbrock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+monocyte+subpopulation+in+human+peripheral+blood.">Identification and characterization of a novel monocyte subpopulation in human peripheral blood.</a> Blood. 74 (7), (1989).</li><li>Venneri, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proangiogenic+TIE2-expressing+monocytes+(TEMs)+in+human+peripheral+blood+and+cancer.">Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.</a> Blood. 109 (12), 5276-5285 (2007).</li><li>Bradfield, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAM-C+regulates+unidirectional+monocyte+transendothelial+migration+in+inflammation.">JAM-C regulates unidirectional monocyte transendothelial migration in inflammation.</a> Blood. 110 (7), 2545-2555 (2007).</li><li>Schenkel, A. R., Mamdouh, Z., Muller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotion+of+monocytes+on+endothelium+is+a+critical+step+during+extravasation.">Locomotion of monocytes on endothelium is a critical step during extravasation.</a> Nature Immunology. 5 (4), 393-400 (2004).</li><li>Luu, N. T., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+the+different+steps+during+neutrophil+migration+through+cultured+endothelial+monolayers+treated+with+tumour+necrosis+factor-alpha.">Kinetics of the different steps during neutrophil migration through cultured endothelial monolayers treated with tumour necrosis factor-alpha.</a> Journal Vascular Research. 36 (6), 477-485 (1999).</li><li>ibidi GmbH. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Shear Stress and Shear Rates for ibidi &#181;-Slides - Based on Numerical Calculations. , (2014).</li><li>Yang, L., Froio, R. M., Sciuto, T. E., Dvorak, A. M., Alon, R., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICAM-1+regulates+neutrophil+adhesion+and+transcellular+migration+of+TNF-alpha-activated+vascular+endothelium+under+flow.">ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow.</a> Blood. 106 (2), 584-592 (2005).</li><li>Yang, C. -. R., Hsieh, S. -. L., Ho, F. -. M., Lin, W. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoy+receptor+3+increases+monocyte+adhesion+to+endothelial+cells+via+NF-kappa+B-dependent+up-regulation+of+intercellular+adhesion+molecule-1,+VCAM-1,+and+IL-8+expression.">Decoy receptor 3 increases monocyte adhesion to endothelial cells via NF-kappa B-dependent up-regulation of intercellular adhesion molecule-1, VCAM-1, and IL-8 expression.</a> Journal of Immunology. 174 (3), 1647-1656 (2005).</li><li>Wong, D., Dorovini-Zis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+vascular+cell+adhesion+molecule-1+(VCAM-1)+by+human+brain+microvessel+endothelial+cells+in+primary+culture.">Expression of vascular cell adhesion molecule-1 (VCAM-1) by human brain microvessel endothelial cells in primary culture.</a> Microvascular Research. 49 (3), 325-339 (1995).</li><li>Bradfield, P. F., Nourshargh, S., Aurrand-Lions, M., Imhof, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAM+family+and+related+proteins+in+leukocyte+migration+(Vestweber+series).">JAM family and related proteins in leukocyte migration (Vestweber series).</a> Arteriosclerosis Thrombosis and Vascular Biology. 27 (10), 2104-2112 (2007).</li><li>Bradfield, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+JAM-C+Expression+Accelerates+Monocyte-Derived+Cell+Exit+from+Atherosclerotic+Plaques.">Divergent JAM-C Expression Accelerates Monocyte-Derived Cell Exit from Atherosclerotic Plaques.</a> PLoS One. 11 (7), e0159679 (2016).</li></ol>

The authors have no competing financial interests.

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>
	</tbody>
</table>

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 ...

For this method can help answer key question in the field of inflammation and immunology. For example, what are the molecular mechanism of leukocyte adhesion and trans-endothelial migration. The main advantage of this technique is that it allows unlimited use discrimination between transmigration and strong addition of monocytes to endothelial monolayer.Although this method provides information about the recruitment of monocytes on the flow, it can also be adapted to other cells of the hematopoietic systems such as T-cells, B-cells, or derivatives of subpopulation thereof. After washing, label the human umbilical vein endothelial cells or HUVEC with 30 microliters of 37 degree Celsius M199 medium supplemented with one micromolar CMFDA for 10 minutes at 37 degree Celsius and 5%Carbon dioxide. At the end of the incubation, wash the cells with 150 microliters of complete M199 medium and incubate the culture in 150 microliters of fresh complete M199 medium for another 30 minutes.Then replace the supernatant with 150 microliters of complete M199 medium containing the appropriate experimental supplements and return the slide chamber culture to the incubator for six hours. For human PAN monocyte isolation, dilute a freshly collected blood sample in PBS supplemented with one millimolar EDTA at a one to one ratio and carefully layer 20 ml of the blood solution onto 20 ml of density gradient medium for density gradient separation by centrifugation. Collect the middle peripheral blood mononuclear cell platelet layer into a new 50 ml tube containing 40 ml of PBS supplemented with 1 millimolar EDTA and bring the final volume to 50 ml with additional PBS-EDTA After counting, isolate the monocytes with the PAN monocyte isolation kit according to the manufacturer's instruction.And wash the cells three times in M199 medium supplemented with 0.5%Bovine Serum Albumin, or BSA, to eliminate any traces of EDTA. The quality of monocyte isolation is critical for ensuring robust transmigration and adhesion result. Thus it is important to strictly follow the manufacturer's recommendation for the isolation.Resuspend the pellet at a 6 times 10 to the 6 monocytes per ml concentration in fresh M199 medium supplemented with 0.5%BSA and divide the cells into one 200 microliter aliquot per microcentrifuge tube per recruitment assay. Then place the cells at 37 degree Celsius for 20 minutes before their injection into the flow chamber. To prepare the fluidic system for the analysis, first insert a male luer connector into one end of an 8 cm long 3 mm thick piece of silicone tubing and connect the other end to an inline luer injection set.Then connect the luer connector to one end of a 40 cm long 3 mm thick piece of silicone tubing. Next, the 20 mm syringe to one end of a 1 m long 3mm thick piece of silicone tubing and insert a male luer connector to the other end. Then insert both male luer connectors into a female luer lock coupler and place the free end of the silicone tubing into a reservoir of 37 degree Celsius M199 medium supplemented with 0.5%BSA.Retract the plunger to fill the tubing with flow buffer and secure the syringe on the syringe pump. Then set the pump to the withdraw mode and specify the flow rate. To connect the slide chamber, clamp the silicone tubing around the female luer lock coupler and disconnect the two luer connector males from the coupler to allow them to be connected to the reservoir of the slide.Air bubbles not only disturb the image quality but also induce capillary sheer stress to the cells, leading to cell death. To avoid air bubbles, confirm the correct clamping of the tubing before inserting the luer into the reservoir. Then remove the clamps to confirm that the connection is not leaking and place the slide under the microscope.For imaging of the monocyte recruitment under flow, add 5 microliters of flourescence conjugated anti CD16 antibody and an appropriate nuclear dye to each aliquoted monocytes for a 10 minute incubation at room temperature and collect the cells with a brief centrifugation. Resuspend the pellet in 250 microliters of flow buffer and start the syringe pump. Add 30 microliters of one monocyte suspension to one chamber of the slide and select the 40X objective on the confocal microscope.Activate the appropriate fluorescent lasers and use the chamber with the labeled monocyte to set the acquisition parameters of the confocal microscope Next, select three fields of view within a half centimeter radius for multi position confocal imaging, and set a Z stack to the 10 to 12 micrometer range and the time lapse acquisition to run every one minute. After three minutes of imaging, inject 200 microliters of monocytes through the inline luer injection port and begin imaging the cellular interaction. After at least 30 minutes, stop the imaging and the flow and clamp the tubing to allow it to be disconnected from the slide.To determine the transmigration rate of the cells through the stimulated HUVECs, open image J and count the total number of adherent monocytes in each field to determine the cell count per square millimeter. Then count the number of transmigrated monocytes that are present in the basal plane underneath the endothelial cells as identified by the presence of a black hole zone around the nucleus. A simple way to check the activation status of the HUVECs after stimulation is to visualization of their elongation under inflammatory stress.Time lapse confocal imaging allows visualization of the monocytes at the apical plane where their phenotype can be assessed. Migrating cells undergoing transmigration move to the intercellular cell-to-cell junction before they disappear from the apical plane and reappear in the basal plane. Quantitation of the monocyte recruitment over time confirms that monocyte adhesion is followed by transmigration.The transmigration rate of CD16 positive monocyte is low when the endothelial cell are stimulated with TNF alpha only but increases when the endothelial cells are stimulated with both TNF alpha and vascular endothelial growth factor or VEGFA. HUVEC stimulation with TNF alpha and TNF alpha plus VEGFA induces an increase in the adherence to CD14 negative T, B and NK cells, compare to CD14 positive monocytes. In addition, HUVEC stimulation induces transmigration of the monocyte population only, as T, B and NK cells do not transmigrate under either TNF alpha or TNF alpha plus VEGFA stimulatory conditions.To perform an optimal monocyte recruitment assay, it is important that you plan your experiment in advance and do it at the same day. And when you purify the cells, make sure that your microscope is at 37 degrees so that you do not transfer the cells to different temperature during the experiment. To learn this procedure, all the methods like the profiling of HUVEC cytokine and chemokine expression as well as chemotaxis studies can be performed to answer additional question about the molecular mechanism that sustain the transmigration and adhesion of specific monocyte population.

simultaneous-study-recruitment-monocyte-subpopulations-under-flow

Research

JoVE Journal

Immunology and Infection

6.7K visualizações.  University of Geneva. Pois este método pode ajudar a responder a perguntas-chave no campo da inflamação e imunologia. Por exemplo, quais são os mecanismos moleculares de adesão leucócito e migração trans-endotelial. A principal vantagem dessa técnica é que ela permite a discriminação de uso ilimitado entre transmigração e forte adição de monócitos à monocamadas endotelial.Embora este método forneça informações sobre o recrutamento de monócitos no fluxo, ele também pode ser adaptado a...