The authors gratefully acknowledge the work of Prof. William Muller and Dr. Clare Westhorpe for their key role in development of earlier iterations of this model. The authors would also like to thank the AMREP Flow Cytometry core for the sorting of monocyte subsets and the Alfred Hospital Infectious Disease Unit clinical research nurses for the recruitment of HIV+ individuals for some studies. The authors gratefully acknowledge the contribution to this work of the Victoria Operational Infrastructure Support Program received by the Burnet Institute. TAA is supported by an RMIT University Vice-Chancellor&#39;s Postdoctoral Fellowship. This work was supported by NHMRC project grant 1108792 awarded to AJ and AH. TK is supported by NIH grants NIH K08AI08272, NIH/NCATS Grant # UL1TR000124.

NOTE: All experiments using human biological samples were performed with ethics approval from the Alfred Hospital Human Ethics Committee, Melbourne. All experiments were performed in Class II Biosafety cabinets unless specified. &#34;Prewarmed&#34; refers to reagents warmed to 37 &#176;C in a waterbath.
1. Preparation of Type I Fibrous Collagen Gels: Day 1
<ol>
	<li>Prepare polymerized collagen gels by sequentially adding and mixing 35.7 mM NaOH, 0.71 x M199, 4.58 mM acetic acid and 1.71 mg/...

<ol>
	<li>Napoli, C., 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=Fatty+streak+formation+occurs+in+human+fetal+aortas+and+is+greatly+enhanced+by+maternal+hypercholesterolemia.+Intimal+accumulation+of+low+density+lipoprotein+and+its+oxidation+precede+monocyte+recruitment+into+early+atherosclerotic+lesions.">Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions.</a> J Clin Invest. 100 (11), 2680-2690 (1997).</li><li>Woollard, K. J., Geissmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+in+atherosclerosis:+subsets+and+functions.">Monocytes in atherosclerosis: subsets and functions.</a> Nat Rev Cardiol. 7 (2), 77-86 (2010).</li><li>Xu, L., 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=Foamy+Monocytes+Form+Early+and+Contribute+to+Nascent+Atherosclerosis+in+Mice+With+Hypercholesterolemia.">Foamy Monocytes Form Early and Contribute to Nascent Atherosclerosis in Mice With Hypercholesterolemia.</a> Arterioscler Thromb Vasc Biol. 35 (8), 1787-1797 (2015).</li><li>Jackson, W. D., Weinrich, T. W., Woollard, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very-low+and+low-density+lipoproteins+induce+neutral+lipid+accumulation+and+impair+migration+in+monocyte+subsets.">Very-low and low-density lipoproteins induce neutral lipid accumulation and impair migration in monocyte subsets.</a> Scientific Reports. 6, 20038 (2016).</li><li>Angelovich, T. A., Hearps, A. C., Jaworowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation-induced+foam+cell+formation+in+chronic+inflammatory+disease.">Inflammation-induced foam cell formation in chronic inflammatory disease.</a> Immunol. Cell. Biol. 93 (8), 683-693 (2015).</li><li>Llodr&#225;, 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=Emigration+of+monocyte-derived+cells+from+atherosclerotic+lesions+characterizes+regressive,+but+not+progressive,+plaques.">Emigration of monocyte-derived cells from atherosclerotic lesions characterizes regressive, but not progressive, plaques.</a> Proc. Natl. Acad. Sci. 101 (32), 11779-11784 (2004).</li><li>Getz, G. S., Reardon, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Atherosclerosis.">Animal Models of Atherosclerosis.</a> Arterioscler. Thromb. Vasc. Biol. 32 (5), 1104-1115 (2012).</li><li>Meir, K. S., Leitersdorf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis+in+the+Apolipoprotein+E-Deficient+Mouse.+A+Decade+of+Progress.">Atherosclerosis in the Apolipoprotein E-Deficient Mouse. A Decade of Progress.</a> Arterioscler. Thromb. Vasc. Biol. 24 (6), 1006-1014 (2004).</li><li>Hilgendorf, I., Swirski, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+a+difference:+Monocyte+Heterogeneity+in+Cardiovascular+Disease.">Making a difference: Monocyte Heterogeneity in Cardiovascular Disease.</a> Curr. Atheroscler. Rep. 14 (5), 450-459 (2012).</li><li>Rogacev, K. 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=Lower+Apo+A-I+and+Lower+HDL-C+Levels+Are+Associated+With+Higher+Intermediate+CD14++CD16++Monocyte+Counts+That+Predict+Cardiovascular+Events+in+Chronic+Kidney+Disease.">Lower Apo A-I and Lower HDL-C Levels Are Associated With Higher Intermediate CD14++CD16+ Monocyte Counts That Predict Cardiovascular Events in Chronic Kidney Disease.</a> Arterioscler. Thromb. Vasc. Biol. 34 (9), 2120-2127 (2014).</li><li>Rogacev, K. 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=CD14++CD16++Monocytes+Independently+Predict+Cardiovascular+Events:+A+Cohort+Study+of+951+Patients+Referred+for+Elective+Coronary+Angiography.">CD14++CD16+ Monocytes Independently Predict Cardiovascular Events: A Cohort Study of 951 Patients Referred for Elective Coronary Angiography.</a> J. Am. Coll. Cardiol. 60 (16), 1512-1520 (2012).</li><li>Justus, C. R., Leffler, N., Ruiz-Echevarria, M., Yang, L. V. <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+Cell+Migration+and+Invasion+Assays.">In vitro Cell Migration and Invasion Assays.</a> J. Vis. Exp. (88), e51046 (2014).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chemotactic+Effect+Of+Mixtures+Of+Antibody+And+Antigen+On+Polymorphonuclear+Leucocytes.">The Chemotactic Effect Of Mixtures Of Antibody And Antigen On Polymorphonuclear Leucocytes.</a> J. Exp. Med. 115 (3), 453-466 (1962).</li><li>Westhorpe, C. L. V., 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=Endothelial+cell+activation+promotes+foam+cell+formation+by+monocytes+following+transendothelial+migration+in+an+in+vitro+model.">Endothelial cell activation promotes foam cell formation by monocytes following transendothelial migration in an in vitro model.</a> Exp. Mol. Pathol. 93, 220-226 (2012).</li><li>Quinn, M. T., Parthasarathy, S., Fong, L. G., Steinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidatively+modified+low+density+lipoproteins:+a+potential+role+in+recruitment+and+retention+of+monocyte/macrophages+during+atherogenesis.">Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis.</a> Proc. Natl. Acad. Sci. 84 (9), 2995-2998 (1987).</li><li>Henriksen, T., Mahoney, E. M., Steinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+macrophage+degradation+of+biologically+modified+low+density+lipoprotein.">Enhanced macrophage degradation of biologically modified low density lipoprotein.</a> Arterioscler. Thromb. Vasc. Biol. 3 (2), 149-159 (1983).</li><li>Parthasarathy, S., Raghavamenon, A., Garelnabi, M. O., Santanam, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+Low-Density+Lipoprotein.">Oxidized Low-Density Lipoprotein.</a> Methods Mol. Biol. 610, 403-417 (2010).</li><li>Muller, W. A., Weigl, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-selective+transendothelial+migration:+dissection+of+the+binding+and+transmigration+phases+by+an+in+vitro+assay.">Monocyte-selective transendothelial migration: dissection of the binding and transmigration phases by an in vitro assay.</a> J. Exp. Med. 176 (3), 819-828 (1992).</li><li>Maisa, 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=Monocytes+from+HIV-infected+individuals+show+impaired+cholesterol+efflux+and+increased+foam+cell+formation+after+transendothelial+migration.">Monocytes from HIV-infected individuals show impaired cholesterol efflux and increased foam cell formation after transendothelial migration.</a> AIDS. 29 (12), 1445-1457 (2015).</li><li>Angelovich, T. 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=Ex+vivo+foam+cell+formation+is+enhanced+in+monocytes+from+older+individuals+by+both+extrinsic+and+intrinsic+mechanisms.">Ex vivo foam cell formation is enhanced in monocytes from older individuals by both extrinsic and intrinsic mechanisms.</a> Exp. Gerontol. 80, 17-26 (2016).</li><li>Westhorpe, C. L. V., 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=Effects+of+HIV-1+infection+in+vitro+on+transendothelial+migration+by+monocytes+and+monocyte-derived+macrophages.">Effects of HIV-1 infection in vitro on transendothelial migration by monocytes and monocyte-derived macrophages.</a> J. Leukoc. Biol. 85 (6), 1027-1035 (2009).</li><li>Furie, M. B., Cramer, E. B., Naprstek, B. L., Silverstein, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+endothelial+cell+monolayers+that+restrict+the+transendothelial+passage+of+macromolecules+and+electrical+current.">Cultured endothelial cell monolayers that restrict the transendothelial passage of macromolecules and electrical current.</a> J. Cell Biol. 98 (3), 1033-1041 (1984).</li><li>M&#252;ller, A. M., 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=Expression+of+the+Endothelial+Markers+PECAM-1,+vWf,+and+CD34+in+Vivo+and+in+Vitro.">Expression of the Endothelial Markers PECAM-1, vWf, and CD34 in Vivo and in Vitro.</a> Exp. Mol. Pathol. 72 (3), 221-229 (2002).</li><li>Kelesidis, T., 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=Predictors+of+impaired+HDL+function+in+HIV-1+infected+compared+to+uninfected+individuals.">Predictors of impaired HDL function in HIV-1 infected compared to uninfected individuals.</a> J. Acquir. Immune Defic. Syndr. , (2017).</li><li>Rogacev, K. 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=Monocyte+heterogeneity+in+obesity+and+subclinical+atherosclerosis.">Monocyte heterogeneity in obesity and subclinical atherosclerosis.</a> European Heart Journal. 31 (3), 369-376 (2010).</li><li>Gacka, M., 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=Proinflammatory+and+atherogenic+activity+of+monocytes+in+Type+2+diabetes.">Proinflammatory and atherogenic activity of monocytes in Type 2 diabetes.</a> J. Diabetes Complications. 24 (1), 1-8 (2010).</li><li>Rogacev, K. 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=CD14++CD16++monocytes+and+cardiovascular+outcome+in+patients+with+chronic+kidney+disease.">CD14++CD16+ monocytes and cardiovascular outcome in patients with chronic kidney disease.</a> Eur. Heart J. 32 (1), 84-92 (2011).</li></ol>

The protocol described here offers a versatile and physiologically relevant method for assessing the atherogenicity of monocytes from human clinical cohorts, by combining both monocyte transmigration and foam cell formation. This model offers advantages over alternative methods of foam cell formation as it takes into account the effect of monocyte transmigration on foam cell formation and allows the measurement of reverse transmigration6 in addition to the inherent propensity of monocytes to matur...

Monocyte transmigration is a crucial step in the development of atherosclerotic plaque that may lead to thrombosis, stroke and myocardial infarction. Atherosclerotic plaques develop from fatty streaks, generally present at sites of low oscillatory blood flow in medium to large arteries, where deposited lipid contributes to endothelial activation and localized inflammation1. Monocytes are recruited to endothelial cells in fatty streaks via monocyte chemotactic proteins (such as CCL2) and transmigrate into the intima2. Following transmigration, monocytes may form atherogenic, lipid-laden macrophages called foam cells as a consequence of lipid uptake, lipid synthesis, down-regulation of cholesterol efflux or a combination of the above factors. Monocytes may also accumulate lipids in the circulation and have a &#39;foamy&#39; phenotype, possibly predisposing cells for foam cell formation3,4. Foam cells are the defining feature of fatty streaks and early-stage atherosclerotic plaques and their formation is influenced by both lipid and inflammatory mediators5. Alternatively, monocytes have the ability to reverse transmigrate from the artery into the bloodstream6, thereby removing lipid from the intima and acting to maintain the health of the artery.
Determining the propensity of monocytes to transmigrate across arterial endothelium and form foam cells in the intima, or to reverse transmigrate and carry lipid out of the plaque, is a key requirement for understanding the role of monocyte activation in increasing atherosclerotic risk. Mouse models of CADs such as atherosclerosis are important in elucidating real-time in vivo information on fatty streak/atherosclerotic plaque development. However, these models require a genetic alteration of the natural cholesterol processing abilities of these animals usually coupled with drastic alterations in diet (such as the ApoE-/- Western-type diet model)7,8, thereby, inducing non-physiological accumulation of circulating lipid levels which drive plaque development. These models may have limited relevance to chronic inflammatory human conditions such as HIV infection which are not associated with increased circulating cholesterol or low-density lipoprotein (LDL) levels. Furthermore, differences in monocyte biology between humans and mice make the testing of immunological questions regarding the relevance of subpopulations of monocytes (such as intermediate monocytes (CD14++CD16+))9 difficult. This is important when studying the mechanisms driving cardiovascular disease as intermediate monocyte counts independently predict cardiovascular events10,11. While assays exist to sequentially measure either monocyte transmigration or foam cell formation in isolation, no in vitro assay has been validated for quantifying both aspects of early atherogenesis using the same cells from clinical cohorts. Transwell models utilize a modified Boyden two-chamber system whereby cells are loaded into the top chamber and transmigrate across a porous plastic barrier or cell monolayer into a lower chamber that typically contains media with chemoattractant12,13. Whilst widely used for analyzing leukocyte transmigration, these models do not generally incorporate a layer representing the intima, resulting in transmigrated cells migrating into solution, and do not allow for the measurement of foam cell formation or reverse transmigration of the same cells. Conversely, models of foam cell formation do not account for any transmigratory-induced changes to monocytes or effects of endothelial activation which is known to contribute to foam cell formation14. Furthermore, these systems induce foam cell formation from macrophages adhered to cell culture plates by the addition of saturating concentrations of exogenous oxidized low-density lipoprotein (oxLDL)15,16, a key inducer of foam cell formation. LDL used in these models is often oxidized by non-physiologically-relevant processes such as CuSO4 treatment17, therefore, questioning the physiological importance of studies using these models.
Here we describe an assay that quantifies monocyte transmigration and foam cell formation of the same cells which does not require the addition of exogenous oxLDL, thus better modelling the role of monocytes in foam cell formation. This model was originally developed by Professor William Muller (Northwestern University, Chicago)18, and has been further refined in our laboratory to assess ex vivo the atherogenicity of monocytes isolated under non-activating conditions from individuals with underlying inflammatory conditions accompanying diseases such as HIV infection19 as well as ageing20, that are associated with an increased risk of atherosclerosis. This model also provides a platform for answering basic biological questions regarding the propensity of different monocyte subsets to form foam cells20, the influence of endothelial activation by cytokines such as TNF on foam cell formation14, and the migratory properties of monocytes such as the depth and speed of transmigration in gels19. Furthermore, monocyte transmigration and foam cell formation can be quantified using standard microscopy, live cell imaging, flow cytometry and imaging flow cytometry, therefore, providing a versatile method to evaluate the role of monocytes in atherogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="1187">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Gel preparation reagents</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>221465-500G</td>
			<td>0.1 M NaOH diluted in H20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">10X M199</td>
			<td>Sigma-Aldrich</td>
			<td style="width:111px;">M0650</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">AcCOOH</td>
			<td>Sigma-Aldrich</td>
			<td>695092-100ML</td>
			<td>20 mM Acetic acid diluted in H20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cultrex Bovine Collagen I</td>
			<td>R&#38;D Systems</td>
			<td>3442-050-01</td>
			<td>Type I Fibrous Collagen</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">M199</td>
			<td>Life Technologies</td>
			<td>11150-059</td>
			<td style="width:595px;">M199 media containing Earle&#39;s salts, L-glutamine and 2.2 g/L Sodium Bicarbonate. 
			Media supplemented with 100 &#181;g/mL L-glutamine&#160; and 100 U/mL penicillin/streptomycin&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">M20</td>
			<td></td>
			<td></td>
			<td style="width:595px;">Supplemented M199 containing 20% heat-inactivated pooled or donor serum. 
			Individual lipid species such as LDL can be added to M199.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">HUVEC</td>
			<td></td>
			<td></td>
			<td style="width:595px;">Primary human umbilical cord endothelial cells (HUVEC) can be isolated from umbilical cords donated with informed consent and ethics approval. Isolated HUVEC may also be purchased commercially.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human coronary artery endothelial cells</td>
			<td></td>
			<td></td>
			<td style="width:595px;">Primary human coronary artery endothelial cells can be isolated from arteries donated with informed consent and ethics approval. Isolated cells may also be purchased commercially.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>BDH Merck</td>
			<td>10093.5V</td>
			<td>Ethylenediaminetetraacetic acid (EDTA) - 0.5 M, pH 8.0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889-500G</td>
			<td>Ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA) - 1 mM, pH 8.0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.05% trypsin EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>0.05% trypsin/0.53 EDTA (1X)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>L-glutamine (200 mM)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Penicillin/streptomycin (10,000 units/mL Penicillin and 10,000 &#181;g/mL Streptomycin)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">New born calf serum</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:111px;">16010-142</td>
			<td style="width:595px;">New born calf serum: New Zealand origin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F1056-1MG</td>
			<td>Fibronectin - 50 &#181;g/mL aliquots prepared and stored</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">PBS (1X)</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (10X): Dilute to 1X with sterile H20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.1% TNF</td>
			<td>Gibco</td>
			<td>PHC3015</td>
			<td>Recombinant human TNF - Reconstituted in H20 and stored in 10 &#181;g/mL aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low-density lipoprotein</td>
			<td>Merck Millipore</td>
			<td>LP2-2MG</td>
			<td>Low-density lipoprotein (LDL)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Microscopy</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1 or 2% formaldehyde</td>
			<td>Polysciences</td>
			<td>4018</td>
			<td>1 or 2% formaldehyde diluted with sterile H20</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">50% and 78% methanol</td>
			<td>Ajax Finechem</td>
			<td>318-2.5L GL</td>
			<td>50% or 78% v/v methanol, diluted with H20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oil Red O stain</td>
			<td>Sigma-Aldrich</td>
			<td>O0625-25G</td>
			<td>Dilute to 2 mg/mL in 22% 1M NaOH and 78% (v/v) methanol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope slides</td>
			<td>Mikro-Glass</td>
			<td>S41104AMK</td>
			<td style="width:595px;">Twin frosted 45 degree ground edge microscope slides (25 X 76 mm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover slips</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>MENCS224015GP</td>
			<td>22 x 40 mm #1.5 size glass cover slips</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double-sided tape</td>
			<td>3M Scotch</td>
			<td>4011</td>
			<td>Super strength exterior mounting tape (25.4 mm x 1.51 m)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Giemsa stain</td>
			<td>Merck Millipore</td>
			<td>1.09204.0500</td>
			<td>Giemsa&#39;s azur eosin methylene blue solution (dilute stock 1:10 in H20)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Hole punch</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;">Hand-held single hole punch (6.35 mm punch)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Flow cytometry</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase D</td>
			<td>Roche Diagnostics</td>
			<td>11088858001</td>
			<td>Collagenase D diluted in M199 media to 1 mg/mL&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 &#181;m nylon mesh capped polystyrene FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>35 &#181;m nylon mesh capped polystyrene FACS tubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live/Dead Fixable Yellow Dead Cell stain</td>
			<td>Life Technologies</td>
			<td>L34959</td>
			<td>Live/Dead Fixable Yellow Dead Cell stain</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">FACS wash</td>
			<td></td>
			<td></td>
			<td>Prepare by mixing 1 X PBS-, 2 mM EDTA and 1% New born calf serum</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Plasticware</td>
			<td style="width:128px;"></td>
			<td style="width:111px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plate</td>
			<td>Nunclon</td>
			<td>167008</td>
			<td>Delta surface flat-bottomed 96 well plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cm Petri Dish</td>
			<td>TPP</td>
			<td>93100</td>
			<td>Sterile 10 cm Petri dish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td>Eppendorf tubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco Scientific</td>
			<td>222-20S</td>
			<td>Sterile transfer pipette (1 mL, large bulb)</td>
		</tr>
	</tbody>
</table>

quantification of monocyte transmigration and foam cell formation from individuals with chronic inflammatory conditions

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

Quantifying monocyte transmigration
Monocytes are added to the model as described in Figure 1, and six gels are prepared for each condition. Monocytes for 6 gels per donor (i.e., 5.0 x 104 monocytes per gel 6 gels = 3.0 x 105 monocytes per donor) are resuspended to a final volume of 600 &#181;L of M199 media containing the required serum/isolated lipid...

Watch this Scientific Journal Video about Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions at JoVE.com

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

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

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

The overall goal of this assay is to quantify the capacity for monocytes from human clinical samples to undergo transendothelial migration and foam cell formation. This method can be used to answer key questions in the cardiovascular field such the atherogenic potential of cells from individuals who are at risk of acute coronary artery disease. The major advantage of this technique is that clinical samples can be measured both from fresh whole blood or stored patient cohorts.Furthermore, foam cell formation can be quantified by both microscopy and flow cytometry. First, prepare the polymerized collagen gels from a fresh collagen suspension. To a five-milliliter polystyrene tube, add sodium hydroxide, then 10 times M199, then acidic acid, and finally, add fibrous collagen.Mix this well by pipetting. Next, aliquot 50 microliters into the internal wells of a sterile flat-bottomed 96-well tissue culture plate. In the outer edges of wells, load 200 microliters of one times Dulbecco PBS to prevent desiccation.After two hours of incubation at 37 degrees Celsius, the collagen will polymerize. Then, overlay the gels with 150 microliters of one times supplemented M199 and incubate them until needed for use five days later. To expand stored human umbilical vein endothelial cells, prepare a 25-square-centimeter culture flask with one milliliter of fibronectin solution and incubate the dish for 10 minutes at room temperature.Meanwhile, thaw the stored cells in a water bath to resuspend them in M20. Once the cells are thawed, aspirate any residual fibronectin solution from the dish and plate the cells. Then, culture to confluence by replacing the media after two to three days.The HUVECs should be confluent on day five of culturing. Detach them by aspirating the culture's supernatant from the flask, and then wash away the serum-containing media using two to three milliliters of one times PBS. Then, add five milliliters of diluted trypsin and incubate the cells briefly at room temperature shaking the cells gently until they appear detached.Then, quickly collect them with five-milliliters of M20 and transfer the suspension to a 10-milliliter tube and centrifuged cells. Now, resuspend the cells in M20 at 200, 000 cells per milliliter. Then, aspirate the M199 from the prepared collagen plate and add 100 microliters of HUVECs to each gel.Continue the culture for three days. On day eight of culturing the HUVECs, activate the monolayers prior to adding the monocytes. After aspirating the overlaying media, add 100 microliters of human TNF-alpha solution to each well.Then, incubate the plate at 37 degrees Celsius for four hours. After the incubation, remove the media and wash the gels twice using 100 microliters of M199 per wash. Now, add 50, 000 freshly isolated monocytes or purified monocyte subsets in 100 microliters of M20 to the HUVEC monolayers.After adding the monocytes, incubate the plate for an hour to allow for forward transmigration of the cells. After an hour, collect the non-transmigrated cells in wash solution. Start by pooling two EGTA washes using 100 microliters per well.Then, spin down the combined wash solutions at 300 G for five minutes at four degrees Celsius. Discard the supernatant, and resuspend the cells in 30 microliters of PBS and count the cells to determine the percentage of forward transmigrated cells. Now, wash plate wells once with M199 and add 100 microliters of fresh M20 to the plate wells and incubate the plate for two days.After two days, repeat the procedure for collecting the non-transmigrated cells in EGTA wash solution in order to collect reverse transmigrated cells and proceed with phenotypic analysis of the cells. To phenotypically characterize the gel-bound cells by FACS analysis, digest the cells by adding 100 microliters of 37-degrees-Celsius collagenese D and incubating for 20 minutes at 37 degrees Celsius. Following the incubation, macerate the gels using a 200-microliter pipette tip and incubate them for a further 20 minutes to fully digest the gels.Once fully digested, filter and pool the digested replicate gels through a 35-micron nylon mesh into a FACS tube on ice. Now, wash the filtered cells once with cold FACS wash solution. Centrifuge the cells and resuspend them in 100 microliters of cold wash solution.Then, perform FACS staining and analysis according to the text protocol. To analyze the cells by microscopy, first stain the cells as described in the text protocol. To detach the stained cells, rim the wells using a 21-gauge needle with the bevel facing outwards.Next, prepare slides to mount the gels. Punch two small holes into a strip of double-sided tape and then fix the tape to the slide and remove the protective coating. Next, add a drop of water to each hole in the tape.Then, using tweezers, gently transfer two gels onto the two holes and cover the tape. Finally, carefully attach a coverslip to the tape and proceed with visualization of the cells. Following transmigration, non-transmigrated cells were counted as described.A total of 15, 000 non-transmigrated cells were recovered, and the percentage of forward transmigration was determined to be 95%Following 48 hours of further incubation, 36, 000 reverse transmigrated cells were recovered which amounted to 12.6%reverse transmigration. Staining the gels with oil red O revealed foam cells pointed out by white arrows and macrophages pointed out with black arrows. These monocytes were treated with oxidized LDL during transmigration, a substance used to induce foam cell formation in conventional foam cell induction models.Aggregate cell count data from 12 donors confirmed that the incubation of monocytes with oxidized LDL induced more foam cell formation than when monocytes are incubated with native LDL or M199 media alone. The transmigrated cells were also analyzed by flow cytometry. After excluding dead cells, doublets and CD45 negative HUVECs, the major population of migrated cells was selected and the mean fluorescence intensity of the receptors of interest was compared with the fluorescence minus one or isotype control data.After watching this video, you should have a good understanding of how to use this assay to quantify monocyte transendothelial migration and foam cell formation from human clinical samples. Don't forget that working with human clinical samples can be extremely hazardous and good laboratory technique must always be used.

quantification-monocyte-transmigration-foam-cell-formation-from

Research

JoVE Journal

Immunology and Infection

13.2K vistas.  Burnet Institute. The overall goal of this assay is to quantify the capacity for monocytes from human clinical samples to undergo transendothelial migration and foam cell formation. This method can be used to answer key questions in the cardiovascular field such the atherogenic potential of cells from individuals who are at risk of acute coronary artery disease. The major advantage of this technique is that clinical samples can be measured both from fresh whole blood or stored patient cohorts.Furthermor...