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A flow cytometric method for the analysis of macrophages in the vascular wall

A flow cytometric method for the analysis of macrophages in the vascular wall
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  A  󿬂 ow cytometric method for the analysis of macrophages inthe vascular wall  Jeffrey P. Moore a,1 , Samy Sakkal b,1 , Michelle L. Bullen a , Barbara K. Kemp-Harper a ,Sharon D. Ricardo c , Christopher G. Sobey a , Grant R. Drummond a, ⁎ a Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria, Australia b School of Biomedical Sciences, Victoria University, St Albans, Victoria, Australia c Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia a r t i c l e i n f o a b s t r a c t  Article history: Received 14 January 2013Received in revised form 3 May 2013Accepted 22 July 2013Available online 6 August 2013 Macrophages accumulate in the vascular wall during conditions such as hypertension andhypercholesterolemia and contribute to vascular remodelling. Here we describe a method forthe isolation and subsequent flow cytometric analysisof macrophages from aortas of mice. Cellsuspensions were prepared from thoracic aortas of male C57BL/6J mice by a combination of manual disruption, incubation in enzymatic digestion medium, and passage through a 70  μ  mcell strainer. Flow cytometric analysis of these suspensions revealed a high content of cellswith strong light scattering properties (i.e. SSC hi ) compared with suspensions derived frommouse blood, spleen, thymus or kidney. Unstained aortic cell suspensions also displayed apreponderance of autofluorescence in the B670, V560, V460, B525 and V610 channels of theflow cytometer, suggesting that these channels should be avoided for subsequent flowcytometric analyses, at least for initial gating steps. Thus, aortic preparations were labelledwith an APC-Cy7-conjugated antibody against the pan-leukocyte marker, CD45, as well as anAPC-conjugated antibody against the macrophage-specific antigen, F4/80, as these fluoro-chromesemit inchannels thatdisplayed relatively low levels of auto-fluorescence inourinitialstudies (i.e. R780 and R660). Flow cytometric analysis of labelled aortic preparations revealeda distinct population of CD45 + F4/80 + cells. Importantly, back-gating on this CD45 + F4/80 + cell population showed it to be now virtually devoid of autofluorescence in all remaining openchannels, and hence an appropriate foundation for further detailed analysis of macrophagepolarization using multiple intra- and extra-cellular markers. Furthermore, we demonstratedthat angiotensin II-induced hypertension in C57BL6/J mice, and hypercholesterolemia inapolipoprotein E-deficient mice, each resulted in an approximate doubling of CD45 + F4/80 + cells in the aortic wall, highlighting the utility of our new protocol for studying the impact of disease on macrophage accumulation in the vascular wall.© 2013 Elsevier B.V. All rights reserved. Keywords: Flow cytometryAortaMacrophageAutofluorescenceHypertensionHypercholesterolemi 1. Introduction Cardiovascularriskstates,includinghypertension,hypercho-lesterolemia and diabetes are associated with the accumulationof macrophages in the vascular wall, especially in large conduitvessels such as the aorta and carotid arteries (Chan et al., 2012;Wenzel et al., 2011). These macrophages accumulate in thesubendothelial space and are a potential source of reactiveoxygen species (ROS) and cytokines, which are likely causes of the endothelial dysfunction and vascular inflammation that arehallmarks of hypertension, hypercholesterolemia and diabetes(Harrison et al., 2012). Moreover, macrophages have a propen-sity to engulf extracellular lipoproteins, particularly those that  Journal of Immunological Methods 396 (2013) 33 – 43 ⁎  Correspondingauthorat:DepartmentofPharmacology,MonashUniversity,Clayton,Victoria3800,Australia.Tel.:+61399054869;fax:+61399029500. E-mail address: (G.R. Drummond). 1 These authors contributed equally.0022-1759/$  –  see front matter © 2013 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect  Journal of Immunological Methods  journal homepage:  have undergone oxidative modification, and in doing sobecome transformed into foam cells, one of the majorcellular constituents of atherosclerotic plaques (Ley et al.,2011).To date, most studies investigating the role of macrophagesin vascular disease states have relied on immunohistochemical(IHC) and histological detection techniques (Bush et al., 2000;Clozel et al., 1991). Although such techniques may providevaluable information about the proximity of macrophages toother cells types and their location within the various layers of the vascular wall, the fact that they are generally performedonly on a small number of thin tissue sections means that datamay not be representative of the entire blood vessel understudy. In addition, such limited sampling may not detect cellspresent in very low numbers. IHC and histological techniquesalso have limitations in discerning between subtypes of closelyrelatedcells,forexample,differentmacrophagesubpopulations.Thisisanimportant issuebecauseidentificationofthedifferentsubpopulations of macrophages in the vascular wall in healthversusdiseaseiscriticalforunderstandingtheirrolesinvascularphysiology and pathophysiology (Gordon and Taylor, 2005).Such information will likely yield novel therapeutic strate-gies for minimizing the detrimental actions of macrophages(i.e. pro-oxidant, pro-inflammatory) without compromisingtheir beneficial effects (i.e. immunological, tissue repairing)on the vascular wall.In contrast to IHC and histological techniques, flow cytom-etryallowsrapidsamplingofcellpopulationsinrelativelylargeamountsoftissue(e.g.anentirebloodvessel).Furthermore,thefact that most standard flow cytometric analysers have thecapacity to simultaneously measure 8 or more fluorescentsignals in a single sample, means that cells and their subpop-ulations can be defined on the basis of multiple surface and/orintracellular cell-specific markers.However, in spite of its potential advantages, there areseveral factors that must be addressed before flow cytometrycanbeemployedtoreliablyquantifycellpopulationsinagiventissue.Onesuchfactoristhequalityofthecellsuspensionuponwhich flow cytometric analysis is performed. Specifically,samples should consist of fully dissociated, viable cells thatretain most (if not all) of the features (e.g. surface antigens)that distinguish them from other cell types  in vivo . Forperipheral blood, and for primary and secondary immuneorgans such as the thymus and spleen, both of which are heldtogether by relatively diffuse extracellular matrices, prepara-tionofviablesinglecellsuspensionscanbeachievedeffectivelywithonlyminimalrequirement(ornorequirementatallinthecaseofblood)formanualdisruptionofthetissue(Swirskietal.,2007). However, for tissues with a high content of high tensilestructural proteins such as collagen and elastin, more robusttechniquesmayberequiredtoliberatecells,usuallyinvolvingacombinationofmanualdisruptionandchemicaldigestionwithcollagenase enzymes. Depending on the incubation time withthedigestiveenzymes,suchprotocolsareusuallyveryefficientat dissociating cells, even from tissues with a high content of tough matrix materials (Galkina et al., 2006; Butcher et al.,2011). However, increasing incubation time with collagenasesmaybringwithitareductionincellviability,aswellascleavageof antigens from the surface of cells that could otherwise havebeen usedtocharacterise them. As suchitis critical to optimizedissociation protocols such that an optimum balance betweensingle cell numbers versus cell viability and antigen expressionis achieved.Another important consideration when analysing cell popu-lations by flow cytometry is the ability to reliably detectantigen-positive cells above a background of autofluorescence.Autofluorescence, which refers to the inherent fluorescentpropertiesofacellsuspension,occurswhenendogenouscellularcomponentsbecomeexcitedbytheexcitationsourceoftheflowcytometer (Hulspas et al., 2009). Molecules such as NADPH andflavins are particularly prone to fluorescing, especially whenexcited with low frequency wavelengths of light (e.g. 488 nm)(Monici, 2005). Structural proteins such as collagen and elastinare also highly autofluorescent (Andersson-Engels et al., 1997;Georgakoudi et al., 2002; Tinker et al., 1983) and this can haveobvious implications for subsequent flow cytometric analysis of cell populations in solid tissues. Hence, for flow cytometricdetectionofantigen-positivecellsinanygiventissue,itiscrucialto have a thorough knowledge of its autofluorescent properties,so that appropriate gating strategies and thresholds abovebackground can be employed.Here we describe a method for isolation and subsequentflow cytometric analysis of macrophages from aortas of micethat is associated with a minimum degree of cell death, andvirtually no reduction in the expression levels of leukocyte-and macrophage-specific surface antigens. Furthermore, wehighlight the fact that cell suspensions generated from aortasare highly auto-fluorescent and provide a flow-cytometricgating strategy that allows macrophages to be reliablyidentified despite these high background signals. 2. Materials and methods  2.1. Animals Male C57BL/6J (8 – 19 weeks-old) and apolipoproteinE-deficient (ApoE − /  − ; 26 weeks-old) mice were used in thisstudy. Mice were obtained from the Monash University CentralAnimal Research Facility and all procedures were approved bythe Monash Animal Research Platform (MARP) Animal EthicsCommittee and conducted in compliance with the NationalHealth and Medical Research Council of Australia's (NHMRC)Guidelines for the Ethical and Humane Use of Animals inresearch. Mice were housed at 25 °C on a 12 h light/dark cycleinmicro-isolatorboxes underspecific pathogen-freeconditionsinlittermategroupsofuptofourindividuals.Micehadaccesstofood and water  ad libitum  and were maintained either onnormal chow or a  “ Western ”  diet (22% fat, 0.12% cholesterol;Specialty Feeds, Australia) from the time of weaning.Hypertensionwasinducedin asubgroupofC57BL/6Jmiceby chronic treatment with angiotensin II (Ang II). Theseanimals underwent surgery to implant an osmotic minipump(Alzet Model 2002; Alzet Corp), which delivered either Ang II(0.7 mg/kg/d,  s.c. ) or, in the case of the control animals,vehicle (0.9% saline,  s.c. ), at a constant infusion rate of 0.11  μ  L/h for 14 d. Briefly, mice were anesthetized byisofluorane inhalation (2 – 4% with oxygen via a nose-cone)and a small lateral incision (5 mm) was made in the skin inthe scapula region. A subcutaneous pocket was then made viabluntdissection,intowhichanosmoticminipumpwasinserted.Thewoundwas closedwitha stainlesssteelclipandmicewere 34  J.P. Moore et al. / Journal of Immunological Methods 396 (2013) 33 – 43  allowed to recover on a heating pad (~10 min) before beingreturned to their home boxes.ApoE − /  − mice utilised for hypercholesterolemia studieswere maintained on a  “ Western ”  diet (described above)normal chow diet until 6 weeks of age and then placed on a"Western" diet (described above) for 21 weeks. For mea-surement of total plasma cholesterol levels, blood from wildtype and ApoE − /  − mice, was collected from the inferior venacava into heparinised tubes and plasma isolated via centri-fugation (4000 ×  g   at 4 °C for 10 min). Plasma total choles-terol levels were then determined using a Roche MODULAR 917 enzymatic colorimetric array (Roche Diagnostics, CastleHill, NSW, Australia).  2.2. Blood pressure measurements Bloodpressurewasmeasuredatdays0,3,7,10and14afterosmotic minipump implantation by tail-cuff plethysmographyusing a MC4000 Multichannel System (Hatteras Instruments,USA).Priortothesurgery,allmicehadundergone “ training ” onthe tail cuff system for at least 3 consecutive days.  2.3. Preparation of cell suspensions Mice were killed via CO 2  asphyxiation and a length of thoracic aorta, extending from proximal to the branch-pointof the left subclavian artery to just distal to the point atwhich the vessel passes through the diaphragm, wasexcised. The perivascular adipose tissue was retained on allspecimens. Aortas were placed in a microcentrifuge tubecontaining a digestion cocktail comprising collagenase type IX(125 U/ml),collagenasetypeI – S(450 U/ml)andhyaluronidaseIS (60 U/ml) (Sigma-Aldrich) dissolved in PBS supplementedwith Ca 2+ and Mg 2+ (Galkina et al., 2006; Guzik et al., 2007).Tissues were then subjected to manual disruption for 3 minusing fine scissors and incubated at 37 °C for either 20, 40 or60 min, while being gently agitated on a rocking platform.Following enzymatic digestion, cells were washed by tworounds of centrifugation at 1200 rpm (283 ×  g  ) for 10 minand re-suspension in PBS buffer supplemented with 5 mMEDTA, 1% bovine serum albumin and 0.02% sodium azide.The suspension was then passed through a 70  μ  m cellstrainer (Falcon, BD) to remove undigested tissue/debris,and a small aliquot (10  μ  L) was taken and stained withTrypan blue for assessment of cell numbers and viabilityusing a Countess Cell Counter (Invitrogen, USA).Kidneys,spleens,thymusesandbloodwerealsoremovedfrom a subset of C57BL/6J mice for subsequent flowcytometric analysis. Kidney cell suspensions were preparedby a combination of manual disruption, enzymatic digestion(for 60 min) and passage through a 70  μ  m cell strainer, asdescribed for aortas above. Spleen and thymus cell prepara-tions were either processed using the conventional glass-slidemethod (Hammond et al., 1998) or, to enable direct compar-isons with aorta and kidney preparations, via the samecollagenase-based digestion procedure described above. Final-ly, whole blood samples were mixed with the anticoagulantClexane (400 U/mL) and analysed on the flow cytometerwithout the need for further processing.  2.4. Antibody labelling  A minimum of 5 × 10 5 cells (based on cell counts obtainedabove) from each aorta were incubated with the appropriateantibody cocktail for 25 min at 4 °C protected from light. Cellswere then washed and resuspended in supplemented PBSbufferforimmediateflowcytometricanalysis.Theviabilitydye7-Amino Actinomycin D (7-AAD) was added to each cellsuspension at a final concentration of 5  μ  g/mL, 3 min prior toanalysisontheflowcytometer.Forisotype,fluorescenceminusone (FMO) and single-colour compensation controls, a similarnumber of aortic cells were labelled as described above withappropriate antibody cocktails. Sample data from 2 × 10 5 events were acquired on a LSR II Flow Cytometer (BDBiosciences, USA) using up to 9 fluorescence channels. Datawere exported as FCS3.0 files and analysed using FlowJosoftware7.6.5(TreestarSoftware,USA).Antibodiesandisotypecontrols included APC Cy7 anti-CD45 (clone 30-F11), APC Cy7Rat IgG2b k isotype control (clone RTK4530), PE anti-NK1.1(clone PK136), PE anti-B220 (clone RA3-6B2), PE anti-CD49b(clone HM α 2), PE anti-CD90.2 (clone 30-H12; BioLegend), PEanti-TER119 (clone TER-119; BD Biosciences), APC anti-F4/80(clone BM8), APC Rat IgG2a k isotype control (clone eBR2a;eBioscience) and 7-AAD (Molecular Probes). Aorta Kidney Spleen Thymus Blood FSC      S     S     C Fig. 1.  Flow cytometric analysis of light scattering properties of single cell suspensions derived from aorta, kidney, spleen, thymus and peripheral blood. Note thepreponderance ofcellswith highside scatter (SSC) andlow forwardscatter (FSC)properties inaorticcell suspensions compared withtheother preparations. Alsonote that only the aorta and kidney cell preparations were prepared using enzymatic digestion protocols. All dot plots are presented following doublet exclusion(FSC-height vs FSC-area).35  J.P. Moore et al. / Journal of Immunological Methods 396 (2013) 33 – 43   2.5. Statistical analyses All data are expressed as mean ± S.E.M. unless otherwisespecified. For statistical comparisons involving more than twoexperimental groups, one-way analysis of variance (ANOVA)was used. For comparisons between two data sets, unpaired ttests were employed. For blood pressure measurements, whichinvolved repeated measures on individual animals over differ-entdays,arepeated-measuresANOVAwasperformed.APvalueof less than 0.05 was considered significant. All statisticalanalyses were performed using Graphpad Prism software(version 6.0b). 3. Results and discussion  3.1. Light scattering and auto  󿬂 uorescence properties of aortic cell suspensions The light scattering properties of aortic cell suspensionswere compared with those of cell suspensions derived fromlymphoid organs and blood (Fig. 1). Whereas the vastmajorityofcellspresentinbloodandlymphoidorgansdisplayedlow side scatter properties (SSC low ), aortic cell suspensions bycomparison contained a large number of cells that were SSC hi .Thesecellsarelikelytoincludethestromalcellsoftheaorticwallsuch as endothelial cells, vascular smooth muscle cells, fibro-blasts and adipocytes, which are present in far higher numbersthanleukocytes,atleastunderphysiologicalconditions.Wealsoexaminedthesidescatterpropertiesofacellsuspensionderivedfrom enzymatic digestion of another non-lymphoid organ, thekidney, and obtained a profile that was intermediate betweenthe aorta and lymphoid organs. Like the aorta, the kidney iscomprised of a complex population of leukocytes and stromalcells. However, notably, the kidneys were largely devoid of adipose tissue (removed with the kidney capsule). Given thatadipocytes have previously been described as a SSC hi cell-type(LeandCheng,2009;Leeetal.,2004),thelackofadipocytesmayexplain why there were fewer SSC hi cells present in cellsuspensions derived from the kidney as compared with theaorta.Overall,theseobservationshighlighttheinherentdifficul-ties in identifying leukocytes amongst the high background of heterogeneous, SSC hi stromal cells in tissues such as the aorta. AortaSpleenKidneyThymus Blood A BC DE      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525 Autofluoroescence Fig. 2.  Flow cytometric analysis of autofluorescence in unstained (A) aortic, (B) kidney, (C) splenic, (D) thymic and (E) blood cell suspensions across commonlyused fluorescence channels. Unstained cell suspensions were analysed for autofluorescence using the following bandpass filters: B670 (used on the Y axis in allgraphs); V610,V460, V560, B525,YG585, YG780, R780 and R660. Note thesubstantially higherlevels ofautofluorescence detected inall channels for aortic versusthe other cell suspensions. All dot plots are presented following doublet exclusion (FSC-height vs FSC-area).36  J.P. Moore et al. / Journal of Immunological Methods 396 (2013) 33 – 43  Cells with high light scattering properties (SSC hi and/orforward scatter-high [FSC hi ]) often display a high degree of autofluorescence, which can interfere with efforts to subse-quently detect cell-specific antigens using fluorescently-conjugated antibodies (Hulspas et al., 2009). Therefore,after excluding doublets (FSC-height vs FSC-area), weexamined the autofluorescent properties of unstained aorticcell suspensions in nine fluorescence channels that arecommonly used for flow cytometric analyses. In all cases, B670was used as the Y-axis parameter, as this channel is renownedfor its tendency to exhibit a high degree of autofluorescenceirrespective of the cell type examined (Mitchell et al., 2010).Even with this consideration, the levels of autofluorescencedetectedintheB670channelweresubstantiallyhigherforaorticcell suspensions (Fig. 2A) than for other cell preparations(Fig. 2B – E). Aortic cellsuspensions also exhibitedahighlevelof autofluorescence in all other channels examined (Fig. 2A).Levels of autofluorescence were particularly high when cellswere analysed through the V560, V460, B525 and V610channels (upper panels in Fig. 2A). Although less autofluores-cence wasdetected in channels R660, YG585,R780and YG780(lowerpanelsinFig.2A),thelevelswerestillgreaterthanthoseobserved in cell suspensions derived from the kidney, lym-phoid organs and blood (Fig. 2B – E).To determine whether the enzymatic digestion protocolused for the aorta and kidney preparations may havecontributed to their higher levels of autofluorescence, wenext examined the impact of this preparation method onautofluorescence levels in spleen and thymus preparations.Autofluorescence levels in all channels examined were moreor less identical regardless of whether preparations wereprepared by enzymatic or non-enzymatic means (Fig. 3A – D).These observations suggest that the autofluorescence observedin the aorta and kidney cell suspensions is an intrinsic propertyofthetissuesfromwhichtheyarederived.Hence,thesefindingshighlight the need for well-designed voltage-setting, com-pensation, fluorophore selection and gating strategies whenanalysingleukocytesubsetsinaortic (andkidney) preparations.The series of experiments depicted in Fig. 4 were carriedout on cell preparations labelled with only 7-AAD and anAPC-Cy7-conjugated CD45 antibody. 7-AAD is a fluorescentcompound that binds with high affinity to double-strandedDNA. Because it does not readily penetrate intact plasmamembranes, it only accumulates in dead or dying cells andcan thus be used to eliminate these cells from subsequentflow cytometric analysis. 7-AAD fluoresces most strongly inthe B670 channel which, based on our findings above, couldpotentially pose problems when working with aortic cellsuspensions (Lecoeur et al., 2002; Philpott et al., 1996). Ascan be seen in Fig. 4A, 7-AAD-positive cell populations wereclearly visible in the kidney, spleen and thymus preparations.By contrast, in aortic cell preparations, the 7-AAD-positivecell population was less clearly defined due to the high levelof background fluorescence in the B670 channel (Fig. 4A).Therefore, to ensure that only the 7-AAD-positive cells wereremoved from subsequent analyses, gating was set based onan unstained aortic sample (i.e. as per Fig. 2A).Havingselectedforviablecells,thepan-leukocytemarker,CD45, was then used to distinguish leukocytes from thestromal cells of the aorta. An APC-Cy7-conjugated antibodywas selected for this critical first step in isolating leukocytesbecause this fluorochrome emits in the R780 channel, which, A BC D      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525      B     6     7     0 YG780YG585R660 R780      B     6     7     0 V610V460V560 B525 SpleenThymus Enzymatic Non -enzymatic SpleenThymus Fig. 3.  Flow cytometric analysis of autofluorescence in unstained (A) enzymatically- and (B) non-enzymatically-prepared thymus cell suspensions, and in(C) enzymatically- and (D) non-enzymatically-prepared splenic cell suspensions across commonly used fluorescence channels. Unstained cell suspensions wereanalysed for autofluorescence using the following bandpass filters: B670 (used on the Y axis in all graphs); V610, V460, V560, B525, YG585, YG780, R780 andR660. All dot plots are presented following doublet exclusion (FSC-height vs FSC-area).37  J.P. Moore et al. / Journal of Immunological Methods 396 (2013) 33 – 43
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