Journal of Immunological Methods 396 (2013) 3343

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Journal of Immunological Methods

journal homepage: www.elsevier.com/locate/jim

A ow cytometric method for the analysis of macrophages in the 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 b c

Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria, Australia School of Biomedical Sciences, Victoria University, St Albans, Victoria, Australia 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
Macrophages accumulate in the vascular wall during conditions such as hypertension and hypercholesterolemia and contribute to vascular remodelling. Here we describe a method for the isolation and subsequent flow cytometric analysis of macrophages from aortas of mice. Cell suspensions 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 m cell strainer. Flow cytometric analysis of these suspensions revealed a high content of cells with strong light scattering properties (i.e. SSChi) compared with suspensions derived from mouse blood, spleen, thymus or kidney. Unstained aortic cell suspensions also displayed a preponderance of autofluorescence in the B670, V560, V460, B525 and V610 channels of the flow cytometer, suggesting that these channels should be avoided for subsequent flow cytometric analyses, at least for initial gating steps. Thus, aortic preparations were labelled with an APC-Cy7-conjugated antibody against the pan-leukocyte marker, CD45, as well as an APC-conjugated antibody against the macrophage-specific antigen, F4/80, as these fluorochromes emit in channels that displayed relatively low levels of auto-fluorescence in our initial studies (i.e. R780 and R660). Flow cytometric analysis of labelled aortic preparations revealed a 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 open channels, and hence an appropriate foundation for further detailed analysis of macrophage polarization using multiple intra- and extra-cellular markers. Furthermore, we demonstrated that angiotensin II-induced hypertension in C57BL6/J mice, and hypercholesterolemia in apolipoprotein 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.

Article history: Received 14 January 2013 Received in revised form 3 May 2013 Accepted 22 July 2013 Available online 6 August 2013 Keywords: Flow cytometry Aorta Macrophage Autofluorescence Hypertension Hypercholesterolemi

1. Introduction Cardiovascular risk states, including hypertension, hypercholesterolemia and diabetes are associated with the accumulation

Corresponding author at: Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia. Tel.: +61 3 9905 4869; fax: +61 3 9902 9500. E-mail address: Grant.Drummond@monash.edu (G.R. Drummond). 1 These authors contributed equally. 0022-1759/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2013.07.009

of macrophages in the vascular wall, especially in large conduit vessels such as the aorta and carotid arteries (Chan et al., 2012; Wenzel et al., 2011). These macrophages accumulate in the subendothelial space and are a potential source of reactive oxygen species (ROS) and cytokines, which are likely causes of the endothelial dysfunction and vascular inflammation that are hallmarks of hypertension, hypercholesterolemia and diabetes (Harrison et al., 2012). Moreover, macrophages have a propensity to engulf extracellular lipoproteins, particularly those that

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have undergone oxidative modification, and in doing so become transformed into foam cells, one of the major cellular constituents of atherosclerotic plaques (Ley et al., 2011). To date, most studies investigating the role of macrophages in vascular disease states have relied on immunohistochemical (IHC) and histological detection techniques (Bush et al., 2000; Clozel et al., 1991). Although such techniques may provide valuable information about the proximity of macrophages to other cells types and their location within the various layers of the vascular wall, the fact that they are generally performed only on a small number of thin tissue sections means that data may not be representative of the entire blood vessel under study. In addition, such limited sampling may not detect cells present in very low numbers. IHC and histological techniques also have limitations in discerning between subtypes of closely related cells, for example, different macrophage subpopulations. This is an important issue because identification of the different subpopulations of macrophages in the vascular wall in health versus disease is critical for understanding their roles in vascular physiology and pathophysiology (Gordon and Taylor, 2005). Such information will likely yield novel therapeutic strategies for minimizing the detrimental actions of macrophages (i.e. pro-oxidant, pro-inflammatory) without compromising their beneficial effects (i.e. immunological, tissue repairing) on the vascular wall. In contrast to IHC and histological techniques, flow cytometry allows rapid sampling of cell populations in relatively large amounts of tissue (e.g. an entire blood vessel). Furthermore, the fact that most standard flow cytometric analysers have the capacity to simultaneously measure 8 or more fluorescent signals in a single sample, means that cells and their subpopulations can be defined on the basis of multiple surface and/or intracellular cell-specific markers. However, in spite of its potential advantages, there are several factors that must be addressed before flow cytometry can be employed to reliably quantify cell populations in a given tissue. One such factor is the quality of the cell suspension upon which flow cytometric analysis is performed. Specifically, samples should consist of fully dissociated, viable cells that retain most (if not all) of the features (e.g. surface antigens) that distinguish them from other cell types in vivo. For peripheral blood, and for primary and secondary immune organs such as the thymus and spleen, both of which are held together by relatively diffuse extracellular matrices, preparation of viable single cell suspensions can be achieved effectively with only minimal requirement (or no requirement at all in the case of blood) for manual disruption of the tissue (Swirski et al., 2007). However, for tissues with a high content of high tensile structural proteins such as collagen and elastin, more robust techniques may be required to liberate cells, usually involving a combination of manual disruption and chemical digestion with collagenase enzymes. Depending on the incubation time with the digestive enzymes, such protocols are usually very efficient at 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 collagenases may bring with it a reduction in cell viability, as well as cleavage of antigens from the surface of cells that could otherwise have been used to characterise them. As such it is critical to optimize dissociation protocols such that an optimum balance between

single cell numbers versus cell viability and antigen expression is achieved. Another important consideration when analysing cell populations by flow cytometry is the ability to reliably detect antigen-positive cells above a background of autofluorescence. Autofluorescence, which refers to the inherent fluorescent properties of a cell suspension, occurs when endogenous cellular components become excited by the excitation source of the flow cytometer (Hulspas et al., 2009). Molecules such as NADPH and flavins are particularly prone to fluorescing, especially when excited with low frequency wavelengths of light (e.g. 488 nm) (Monici, 2005). Structural proteins such as collagen and elastin are also highly autofluorescent (Andersson-Engels et al., 1997; Georgakoudi et al., 2002; Tinker et al., 1983) and this can have obvious implications for subsequent flow cytometric analysis of cell populations in solid tissues. Hence, for flow cytometric detection of antigen-positive cells in any given tissue, it is crucial to have a thorough knowledge of its autofluorescent properties, so that appropriate gating strategies and thresholds above background can be employed. Here we describe a method for isolation and subsequent flow cytometric analysis of macrophages from aortas of mice that is associated with a minimum degree of cell death, and virtually no reduction in the expression levels of leukocyteand macrophage-specific surface antigens. Furthermore, we highlight the fact that cell suspensions generated from aortas are highly auto-fluorescent and provide a flow-cytometric gating strategy that allows macrophages to be reliably identified despite these high background signals.

2. Materials and methods 2.1. Animals Male C57BL/6J (819 weeks-old) and apolipoprotein E-deficient (ApoE/; 26 weeks-old) mice were used in this study. Mice were obtained from the Monash University Central Animal Research Facility and all procedures were approved by the Monash Animal Research Platform (MARP) Animal Ethics Committee and conducted in compliance with the National Health and Medical Research Council of Australia's (NHMRC) Guidelines for the Ethical and Humane Use of Animals in research. Mice were housed at 25 C on a 12 h light/dark cycle in micro-isolator boxes under specific pathogen-free conditions in littermate groups of up to four individuals. Mice had access to food and water ad libitum and were maintained either on normal chow or a Western diet (22% fat, 0.12% cholesterol; Specialty Feeds, Australia) from the time of weaning. Hypertension was induced in a subgroup of C57BL/6J mice by chronic treatment with angiotensin II (Ang II). These animals 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 L/h for 14 d. Briefly, mice were anesthetized by isofluorane inhalation (24% with oxygen via a nose-cone) and a small lateral incision (5 mm) was made in the skin in the scapula region. A subcutaneous pocket was then made via blunt dissection, into which an osmotic minipump was inserted. The wound was closed with a stainless steel clip and mice were

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allowed to recover on a heating pad (~10 min) before being returned to their home boxes. ApoE/mice utilised for hypercholesterolemia studies were 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 measurement of total plasma cholesterol levels, blood from wild type and ApoE/ mice, was collected from the inferior vena cava into heparinised tubes and plasma isolated via centrifugation (4000 g at 4 C for 10 min). Plasma total cholesterol levels were then determined using a Roche MODULAR 917 enzymatic colorimetric array (Roche Diagnostics, Castle Hill, NSW, Australia). 2.2. Blood pressure measurements Blood pressure was measured at days 0, 3, 7, 10 and 14 after osmotic minipump implantation by tail-cuff plethysmography using a MC4000 Multichannel System (Hatteras Instruments, USA). Prior to the surgery, all mice had undergone training on the tail cuff system for at least 3 consecutive days. 2.3. Preparation of cell suspensions Mice were killed via CO2 asphyxiation and a length of thoracic aorta, extending from proximal to the branch-point of the left subclavian artery to just distal to the point at which the vessel passes through the diaphragm, was excised. The perivascular adipose tissue was retained on all specimens. Aortas were placed in a microcentrifuge tube containing a digestion cocktail comprising collagenase type IX (125 U/ml), collagenase type IS (450 U/ml) and hyaluronidase IS (60 U/ml) (Sigma-Aldrich) dissolved in PBS supplemented with Ca2+ and Mg2+ (Galkina et al., 2006; Guzik et al., 2007). Tissues were then subjected to manual disruption for 3 min using fine scissors and incubated at 37 C for either 20, 40 or 60 min, while being gently agitated on a rocking platform. Following enzymatic digestion, cells were washed by two rounds of centrifugation at 1200 rpm (283 g) for 10 min and re-suspension in PBS buffer supplemented with 5 mM EDTA, 1% bovine serum albumin and 0.02% sodium azide. The suspension was then passed through a 70 m cell

strainer (Falcon, BD) to remove undigested tissue/debris, and a small aliquot (10 L) was taken and stained with Trypan blue for assessment of cell numbers and viability using a Countess Cell Counter (Invitrogen, USA). Kidneys, spleens, thymuses and blood were also removed from a subset of C57BL/6J mice for subsequent flow cytometric analysis. Kidney cell suspensions were prepared by a combination of manual disruption, enzymatic digestion (for 60 min) and passage through a 70 m cell strainer, as described for aortas above. Spleen and thymus cell preparations were either processed using the conventional glass-slide method (Hammond et al., 1998) or, to enable direct comparisons with aorta and kidney preparations, via the same collagenase-based digestion procedure described above. Finally, whole blood samples were mixed with the anticoagulant Clexane (400 U/mL) and analysed on the flow cytometer without the need for further processing.

2.4. Antibody labelling A minimum of 5 105 cells (based on cell counts obtained above) from each aorta were incubated with the appropriate antibody cocktail for 25 min at 4 C protected from light. Cells were then washed and resuspended in supplemented PBS buffer for immediate flow cytometric analysis. The viability dye 7-Amino Actinomycin D (7-AAD) was added to each cell suspension at a final concentration of 5 g/mL, 3 min prior to analysis on the flow cytometer. For isotype, fluorescence minus one (FMO) and single-colour compensation controls, a similar number of aortic cells were labelled as described above with appropriate antibody cocktails. Sample data from 2 105 events were acquired on a LSR II Flow Cytometer (BD Biosciences, USA) using up to 9 fluorescence channels. Data were exported as FCS3.0 files and analysed using FlowJo software 7.6.5 (Treestar Software, USA). Antibodies and isotype controls included APC Cy7 anti-CD45 (clone 30-F11), APC Cy7 Rat IgG2b k isotype control (clone RTK4530), PE anti-NK1.1 (clone PK136), PE anti-B220 (clone RA3-6B2), PE anti-CD49b (clone HM2), PE anti-CD90.2 (clone 30-H12; BioLegend), PE anti-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

SSC

FSC
Fig. 1. Flow cytometric analysis of light scattering properties of single cell suspensions derived from aorta, kidney, spleen, thymus and peripheral blood. Note the preponderance of cells with high side scatter (SSC) and low forward scatter (FSC) properties in aortic cell suspensions compared with the other preparations. Also note 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).

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A
Autofluoroescence

Aorta

B
B670

Kidney

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V560 B670

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V560

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C
B670

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YG585

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D
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Thymus

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E
B670

R660

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YG780

R660

YG585

R780

YG780

Blood

V560 B670

V460

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V610

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YG585

R780

YG780

Fig. 2. Flow cytometric analysis of autofluorescence in unstained (A) aortic, (B) kidney, (C) splenic, (D) thymic and (E) blood cell suspensions across commonly used fluorescence channels. Unstained cell suspensions were analysed for autofluorescence using the following bandpass filters: B670 (used on the Y axis in all graphs); V610, V460, V560, B525, YG585, YG780, R780 and R660. Note the substantially higher levels of autofluorescence detected in all channels for aortic versus the other cell suspensions. All dot plots are presented following doublet exclusion (FSC-height vs FSC-area).

2.5. Statistical analyses All data are expressed as mean S.E.M. unless otherwise specified. For statistical comparisons involving more than two experimental groups, one-way analysis of variance (ANOVA) was used. For comparisons between two data sets, unpaired t tests were employed. For blood pressure measurements, which involved repeated measures on individual animals over different days, a repeated-measures ANOVA was performed. A P value of less than 0.05 was considered significant. All statistical analyses were performed using Graphpad Prism software (version 6.0b). 3. Results and discussion 3.1. Light scattering and autouorescence properties of aortic cell suspensions The light scattering properties of aortic cell suspensions were compared with those of cell suspensions derived from lymphoid organs and blood (Fig. 1). Whereas the vast

majority of cells present in blood and lymphoid organs displayed low side scatter properties (SSClow), aortic cell suspensions by comparison contained a large number of cells that were SSChi. These cells are likely to include the stromal cells of the aortic wall such as endothelial cells, vascular smooth muscle cells, fibroblasts and adipocytes, which are present in far higher numbers than leukocytes, at least under physiological conditions. We also examined the side scatter properties of a cell suspension derived from enzymatic digestion of another non-lymphoid organ, the kidney, and obtained a profile that was intermediate between the aorta and lymphoid organs. Like the aorta, the kidney is comprised of a complex population of leukocytes and stromal cells. However, notably, the kidneys were largely devoid of adipose tissue (removed with the kidney capsule). Given that adipocytes have previously been described as a SSChi cell-type (Le and Cheng, 2009; Lee et al., 2004), the lack of adipocytes may explain why there were fewer SSChi cells present in cell suspensions derived from the kidney as compared with the aorta. Overall, these observations highlight the inherent difficulties in identifying leukocytes amongst the high background of heterogeneous, SSChi stromal cells in tissues such as the aorta.

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Enzymatic

Non-enzymatic

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B525

V610 B670

V560

V460

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V610

R660

YG585

R780

YG780

R660

YG585

R780

YG780

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 were analysed for autofluorescence using the following bandpass filters: B670 (used on the Y axis in all graphs); V610, V460, V560, B525, YG585, YG780, R780 and R660. All dot plots are presented following doublet exclusion (FSC-height vs FSC-area).

Cells with high light scattering properties (SSChi and/or forward scatter-high [FSChi]) often display a high degree of autofluorescence, which can interfere with efforts to subsequently detect cell-specific antigens using fluorescentlyconjugated antibodies (Hulspas et al., 2009). Therefore, after excluding doublets (FSC-height vs FSC-area), we examined the autofluorescent properties of unstained aortic cell suspensions in nine fluorescence channels that are commonly used for flow cytometric analyses. In all cases, B670 was used as the Y-axis parameter, as this channel is renowned for its tendency to exhibit a high degree of autofluorescence irrespective of the cell type examined (Mitchell et al., 2010). Even with this consideration, the levels of autofluorescence detected in the B670 channel were substantially higher for aortic cell suspensions (Fig. 2A) than for other cell preparations (Fig. 2BE). Aortic cell suspensions also exhibited a high level of autofluorescence in all other channels examined (Fig. 2A). Levels of autofluorescence were particularly high when cells were analysed through the V560, V460, B525 and V610 channels (upper panels in Fig. 2A). Although less autofluorescence was detected in channels R660, YG585, R780 and YG780 (lower panels in Fig. 2A), the levels were still greater than those observed in cell suspensions derived from the kidney, lymphoid organs and blood (Fig. 2BE). To determine whether the enzymatic digestion protocol used for the aorta and kidney preparations may have contributed to their higher levels of autofluorescence, we next examined the impact of this preparation method on autofluorescence levels in spleen and thymus preparations. Autofluorescence levels in all channels examined were more or less identical regardless of whether preparations were

prepared by enzymatic or non-enzymatic means (Fig. 3AD). These observations suggest that the autofluorescence observed in the aorta and kidney cell suspensions is an intrinsic property of the tissues from which they are derived. Hence, these findings highlight the need for well-designed voltage-setting, compensation, fluorophore selection and gating strategies when analysing leukocyte subsets in aortic (and kidney) preparations. The series of experiments depicted in Fig. 4 were carried out on cell preparations labelled with only 7-AAD and an APC-Cy7-conjugated CD45 antibody. 7-AAD is a fluorescent compound that binds with high affinity to double-stranded DNA. Because it does not readily penetrate intact plasma membranes, it only accumulates in dead or dying cells and can thus be used to eliminate these cells from subsequent flow cytometric analysis. 7-AAD fluoresces most strongly in the B670 channel which, based on our findings above, could potentially pose problems when working with aortic cell suspensions (Lecoeur et al., 2002; Philpott et al., 1996). As can be seen in Fig. 4A, 7-AAD-positive cell populations were clearly visible in the kidney, spleen and thymus preparations. By contrast, in aortic cell preparations, the 7-AAD-positive cell population was less clearly defined due to the high level of background fluorescence in the B670 channel (Fig. 4A). Therefore, to ensure that only the 7-AAD-positive cells were removed from subsequent analyses, gating was set based on an unstained aortic sample (i.e. as per Fig. 2A). Having selected for viable cells, the pan-leukocyte marker, CD45, was then used to distinguish leukocytes from the stromal cells of the aorta. An APC-Cy7-conjugated antibody was selected for this critical first step in isolating leukocytes because this fluorochrome emits in the R780 channel, which,

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J.P. Moore et al. / Journal of Immunological Methods 396 (2013) 3343

Aorta

Kidney

Spleen

Thymus

B670

V610

R780

FSC

B670

R660
Fig. 4. Flow cytometric analysis of the viable leukocyte populations present in single cell suspensions derived from aorta, kidney, spleen, and thymus. Panels depicted in Row A highlight the relative difficulty in excluding 7-AAD-positive cells in aortic preparations due to the high levels autofluorescence detected in the B670 channel. Panels in Row B demonstrate the well-defined cell populations detected following exclusion of dead cells in aortic and non-aortic preparations alike using an APC-Cy7-conjugated antibody against the pan-leukocyte marker CD45. In Row C, back-gating onto open channels (in this example R660) reveals that the APC-Cy7-conjugated anti-CD45 antibody effectively eliminates the autofluorescent cells that were initially present in the aortic preparation, opening up the potential for detailed leukocyte subset characterisation using multiple fluorochromes. All dot plots are presented following doublet exclusion (FSC-height vs FSC-area).

in our earlier studies on unstained aortic cell suspensions, displayed a relatively low level of autofluorescence compared to other channels. The dot plots presented in Fig. 4B show clear populations of CD45-positive cells in all the preparations examined. Not surprisingly, the relative proportion of CD45-positive to CD45-negative (i.e. stromal) cells was markedly lower in aorta and kidney (b 1%), as compared with spleen (~ 5060%) and thymus (~ 7080%). Importantly, when the CD45-positive aortic cell population was back-gated on the remaining open channels, autofluorescence was dramatically reduced in all cases, and virtually absent in channels R660, YG585 and YG780 (Fig. 4C; see also Supplementary Fig. 1). Hence, these studies demonstrate the utility of the APC-Cy7-conjugated CD45 antibody for eliminating most of the autofluorescent cell populations of the aortic wall. Furthermore, these studies highlight the R660, YG585 and YG780 (and R780) as the most appropriate

channels for further characterisation of leukocyte subsets in the aorta. The previous information was used to devise a gating strategy to analyse the macrophage population in aortas. In addition to 7-AAD and CD45-labelling, cell preparations were labelled with an F4/80-APC monoclonal antibody for positive detection of macrophages (Hume et al., 1983), as well as with a cocktail of PE-conjugated antibodies against markers of several non-myeloid cell types including: CD49b and NK1.1 (NK and NKT cells); B220 (B cells); CD90 (T cells); and Ter119 (erythroid cells). Isotype and FMO controls were also prepared and used to set thresholds for positive expression of the respective antigens. APC and PE were selected as fluorochromes because they emit in the R660 and YG585 channels, respectively, which were identified as being two of the least autofluorescent channels in our analysis of aortic leukocytes (see Fig. 4C; see also Supplementary Fig. 1). After

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Doublet Exclusion

Dead Cells CD45+

FSC-height

B670

Viable Cells

R780

FSC-area

V610

FSC

Dump Gate

F4/80+

YG585

R780

R660

R780

7-AAD FMO

Dump FMO

F4/80 Isotype Control R660

CD45 Isotype Control FSC

V610

YG585

R780

B670

R780

R780

Fig. 5. Application of flow cytometric gating strategy to quantify macrophage numbers in the aortic wall of mice. Following exclusion of doublets and dead cells, leukocytes were identified using an APC-Cy7-conjugated anti-CD45 antibody. Non-myeloid cells were then excluded from further analysis using a PE-conjugated antibody cocktail against CD49b, NK1.1, B220; CD90 and Ter119, and finally macrophages were identified with an APC-conjugated anti-F4/80 antibody. Isotype or FMO controls for each of the above antibodies were used to determine positive population (see bottom row).

omission of doublets (FSC-H vs FSC-A) and dead cells (7-AAD-positive), the aortic leukocyte population was identified by gating on CD45, as described above (Fig. 5). Next, non-myeloid cells were excluded from further analysis by gating on the PE-negative population (Fig. 5). Finally, the remaining cells were defined as either macrophages or non-macrophage myeloid cells (e.g. dendritic cells and/or granulocytes) based on their positive or negative expression of the F4/80 antigen (Fig. 5). 3.2. Impact of tissue digestion time on cell yield, viability and antigen expression To assess the impact of enzymatic digestion time on leukocyte viability and yield, manually disrupted aortic homogenates were incubated in the collagenase-based digestion cocktail for either 20, 40 or 60 min, stained with 7-AAD and antibodies, and analysed on the flow cytometer. There was a slight trend (albeit non-significant) for the 60 min incubation time to liberate more total leukocytes and macrophages from the aortic wall, than either the 40 or 20 min incubation protocols (Fig. 6A) while varying incubation times appeared to have minimal impact on leukocyte viability (Fig. 6B). Enzymatic

digestion may also result in the total or partial cleavage of certain cell surface proteins. Such cleavage could potentially affect the expression/presentation of epitopes used for antibody-specific identification of leukocytes in flow cytometry. Although CD45 is reported to be relatively resistant to enzymatic cleavage (Gray et al., 2002, 2008), we nevertheless assessed the impact of the different digestion times used herein on expression of this antigen by measuring the median fluorescence intensity of the signal emitted from the cell population detected in the R780 channel. Importantly, varying the incubation time had no impact on the median fluorescence intensity of the signal emitted from the CD45+ cell population. This suggests that there was minimal effect on extracellular presentation of the CD45 antigen, even with the 60 min exposure time (Fig. 6C). Likewise, median fluorescence intensity of the F4/80 signal was not altered by changing the digestion time, again indicating that expression of this antigen was not affected by our digestion protocols (Fig. 6C). Finally, we also found that there was no effect of varying the digestion time on the median fluorescence intensity of the signal emitted from cells detected in the dump gate using the PE-conjugated antibody cocktail (Supplementary Fig. 2). This indicates that the digestion

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A
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Cell Viability CD45

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Antigen Expression
Median Fluorescence Intensity
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J.P. Moore et al. / Journal of Immunological Methods 396 (2013) 3343

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% Viable Cells

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60 40 20 0 0 102 103 104 CD45 105

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Fig. 6. Effect of varying incubation times in a collagenase/hyaluronidase-based digestion medium on (A) total numbers and (B) percentage viability of leukocytes (CD45+) and macrophages (CD45+F4/80+) in aortic cell suspensions. (C) Shows the lack of effect of the varying incubation times on expression levels of the CD45+ and F4/80+, as assessed by median fluorescence intensity (MFI).

Hypertension

A
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170 160 150 140 130 120 110 100 0 3 7 10 14
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Plasma Cholesterol (mmol/L)

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200 150 100 50 0

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ApoE-/-

ApoE-/-

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Fig. 7. Application of the aortic digestion and flow cytometric gating strategy to assess the impact of experimentally-induced cardiovascular risk factors on macrophage numbers in the aortic wall. Panels (A) and (B) illustrate the effect of 14 d of Ang II infusion on systolic BP and accumulation of macrophages (CD45+F4/80+) in the aortic wall of mice, respectively. Panel (C) shows plasma cholesterol levels of hypercholesterolemic ApoE / versus the normocholesterolemic wild type strain. Panel (D) shows macrophage numbers in the aortic wall of hypercholesterolemic ApoE/ versus the normocholesterolemic wild type strain. In B and D, left hand panels show group data (mean S.E.M.; n 6), while right hand panels show representative flow cytometric dot plots. *P b 0.05 by 2-way repeated measures ANOVA or unpaired t test.

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protocol also had minimal effects on presentation of the various antigens used to exclude non-myeloid leukocyte subsets in our gating strategy. 3.3. Application of aortic ow cytometry protocol to detect changes in macrophage numbers during experimentally-induced disease conditions Hypertension and hypercholesterolemia are two major risk factors for the development of atherosclerosis, which is the primary cause of myocardial infarctions and strokes. One of the earliest processes in the development of atherosclerotic plaques is the accumulation of macrophages in the sub-endothelial compartment of large arteries (Libby et al., 2011; Murray and Wynn, 2011). Hence, we used the flow cytometric method detailed above to quantify macrophage accumulation in the aortic wall in experimental models of hypertension and hypercholesterolemia. Ang II is an octa-peptide produced in the kidneys that regulates blood pressure by activating the sympathetic nervous system, promoting Na+/H2O reabsorption in the kidneys, and via direct constrictor effects on the vasculature (Harrison et al., 2011). The treatment of mice with Ang II for 14 d caused a marked elevation in systolic BP compared to saline infusion (Fig. 7A). Consistent with previous findings using alternative approaches such as immunohistochemistry (Bush et al., 2000), 14 d of Ang II-induced hypertension was associated with a significant increase (~2-fold) in macrophage numbers in the aortic wall (Fig. 7B), along with a similar change in total leukocyte numbers (i.e. a 2-fold increase from 722 111 to 1433 316 CD45+ cells per 2 105 events in normotensive versus hypertensive aortas, respectively; P b 0.05). Likewise, in 26 week-old ApoE/ mice, which are genetically pre-disposed to the development of hypercholesterolemia, there was a significant increase in plasma cholesterol (Fig. 7C) along with a 2-fold increase in macrophage numbers in the aortic wall, which is also consistent with previous reports (Glass and Witztum, 2001; Judkins et al., 2010) (Fig. 7D). In addition, compared to wild-type mice, there was a trend towards an increase in total leukocyte numbers in the aortic wall of ApoE/ mice (1100 226 to 1360 279 CD45+ cells per 2 105 events, respectively). 3.4. Concluding remarks Conditions such as hypertension and hypercholesterolemia are associated with an influx of leukocytes into the arterial wall; a process that likely underpins vascular sequelae such as remodelling, fibrosis and formation of atherosclerotic plaques. Hence, a better understanding of the leukocyte subsets that are present, and their activation state in diseased versus healthy blood vessels may give insights into novel therapeutic interventions to prevent/treat vascular disease. In the present study we have described a protocol that allows robust and reproducible flow cytometric analysis of leukocyte populations in the aortic wall of mice under steady state conditions and also in relevant models of hypertension and hypercholesterolemia. We have shown that enzymatic digestion of aortic homogenates for up to 60 min yields single cell suspensions containing a high percentage of viable cells. Moreover, this digestion protocol appears to have minimal impact on the expression levels of key

cell-specific antigens on the surface of the leukocytes within the suspension. We have also demonstrated that the high content of stromal cells (i.e. smooth muscle, fibroblasts, adipocytes) present in aortic cell preparations makes them highly autofluorescent, especially when excited with wavelengths in the violet or blue region of the light spectrum. However, by using the flow cytometric gating strategy devised herein, which avoids these short wavelength excitation channels for the initial gating steps, the impact of this autofluorescence on detection of antigen-positive leukocytes can be largely avoided, highlighting flow cytometry as a powerful tool for future studies aimed at investigating the pathogenesis of vascular disease. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jim.2013.07.009. Conicts of interest None. Acknowledgements This work was supported by project grants from the NHMRC (1041326) and the National Heart Foundation of Australia (GM11M5825). GRD and CGS are supported by Senior Research Fellowships from the NHMRC, while JM and MB receive PhD stipends from Monash University. None of the funding sources outlined above had any role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. References
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