Name: Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation (Video) | JoVE Journal
Uploaded: 2017-04-03T14:00:00.000+00:00
Duration: 7 min 46 s
Description: Learn to isolate and characterize macrophages from liver tissue affected by obesity-related inflammation. This optimized protocol enables efficient single-cell suspension for immunology and metabolic disorder research.

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5 minutes

We would like to thank the Flow Cytometry Core Facility at The Pennsylvania State University Millennium Science Complex.

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</ol>

Authors have no conflict of interests to disclose.

Diet-induced metabolic disorder models that mimic co-morbidities such as atherosclerosis, simple steatosis, steatohepatitis and type 2 diabetes are extensively used to better understand the underlying molecular mechanisms of disease progression. Collagenase dependent digestion is often used to dissociate tissues to liberate cells from the extracellular matrix (ECM)16,27. Enzymes such as collagenase disrupt collagen which provides structural support for neighborin...

Mouse models have been extensively used to study the dynamics of human diseases. Proper isolation of tissue resident cells from mice in a diseased state can provide a platform for understanding the molecular and cellular contributions to the pathogenesis of a disease1. One disorder that is of critical importance is obesity. The incidence of obesity continues to rise worldwide in parallel with insulin resistance and type 2 diabetes mellitus, cardiovascular disease, and fatty liver disease2,3. Excessive nutrient consumption is further skewed by decreased physical activity triggering altered signals emanating from adipose tissue, which can alter the cellular milieu of other peripheral tissues such as the aorta and liver4. Such disruption of metabolic homeostasis results in a chronic low grade systemic inflammation5.
Classical activation of macrophages resident to the aorta and liver as well as their recruitment to white adipose tissue (WAT) has been shown to not only initiate dysregulation of metabolic signals but also sustain inflammation6,7. The phenotypic and functional heterogeneity of macrophages is strongly associated with the pathogenesis of obesity related co-morbidities7. The dynamic plasticity in macrophage polarization allows for these cells to exhibit a range of activated phenotypes that coordinate the progression and resolution of inflammation8. While classically activated (M1) macrophages are implicated in the propagation of inflammation, alternatively activated (M2) macrophages have been associated with resolution and tissue repair9,10.
As the body undergoes metabolic stress, white adipose tissue abnormally accumulates. The expanded adipose tissue attracts and retains inflammatory cells that profoundly alter normal adipocyte function to promote insulin resistance, hyperglycemia and ultimately type 2 diabetes mellitus, insulin resistance or hyperglycemia11,12. In parallel, white adipose tissue remodels in response to inflammatory signals released by infiltrated classically activated (M1) adipose tissue macrophages (ATMs)13,14. This multi-cellular organ exerts a cascade of signals that derails the normal function of other body organs such as the aorta and liver4.
The liver is a metabolic powerhouse that adapts in response to stimuli originating from nearby dysregulated WAT15. Hepatic macrophages or Kupffer cells, in response to metabolic changes, secrete inflammatory cytokines that transform both parenchymal and non-parenchymal cell phenotype and promote tissue remodeling. Hepatic lipid accumulation, inflammation, excessive extracellular matrix deposits, necrosis and eventual function loss follows the inflammatory insults contributing to the wide spectrum of liver damage associated with non-alcoholic fatty liver disease16,17,18.
In parallel to compromised WAT and liver function, large arteries accumulate lipids within the arterial wall as the body undergoes chronic metabolic stress19. Arterial lipid accumulation triggers the secretion of chemokines by activated endothelial cells and subsequent recruitment of monocytes20. Once recruited, monocytes proliferate, differentiate, ingest lipoproteins and become foam cells. Atherogenesis is initiated and sustained by the pro-inflammatory activity of recruited and tissue resident lipid-laden macrophages. Succumbing to the extracellular and intracellular stress signals relayed in this atherogenic microenvironment, these macrophages then engage in an apoptotic signaling cascade. As these foam cells die, they contribute their lipid filled contents to the necrotic core of the lesion, which then leads to plaque rupture, myocardial infarction, and stroke.
Collectively, the heterogeneity of macrophage phenotypes in part orchestrates the obesity induced by inflammatory changes observed in dysregulated tissues such as WAT, liver and aorta8,21. Characterization of recruited and tissue resident macrophages could provide insight into potential molecular targets that manipulate macrophage phenotype1. To effectively characterize macrophages from obesity-induced inflamed tissues, a single-cell suspension can be obtained through enzymatic digestion. Such dissociation protocols must be effective in sufficiently degrading connective tissue while minimizing immune cell death and providing optimal cell yield. The enzyme mixture is dependent on the type of tissue and its structural make up. Resilient tissues such as the aorta requires stronger enzymatic activity, as compared to the liver and WAT, to achieve tissue dissociation. From the single cell suspension, tissue resident macrophages can be unambiguously characterized or isolated for further downstream analyses such as transcriptional profiling.
Here a tissue-specific protocol is described that uses collagenase-dependent tissue digestion and polychromatic flow cytometry to effectively isolate and characterize tissue-resident macrophages obtained from traditional diet induced obesity, atherosclerosis, simple steatosis and steatohepatitis mouse models. Simultaneous staining of cell surface markers with antibodies against leukocyte- (CD45 and/or CD11b) and macrophage- (F4/80) specific antigens is often used to identify macrophage populations22. Fluorescence-activated cell sorting (FACS) is a powerful strategy used to sort these identified populations at high purity. The sorted population can then be evaluated for phenotype specific gene profiles using downstream molecular analysis (such as quantitative real time polymerase chain reaction)23. Although standard flow cytometry and flow cytometry-based cell sorting are powerful tools in distinguishing macrophages within a vastly heterogeneous cell suspension, the former protocols must be optimized to ensure successful output. In this study, protocols that effectively isolate and characterize viable tissue specific macrophages are described; more importantly, this study provides crucial insight into technical issues that often arise, as well as proactive and trouble-shooting strategies to prevent and/or resolve them.

<table><tbody><tr><td>26 G x 5/8 in Needles</td><td>BD</td><td>305115</td><td></td></tr><tr><td>23 G x 0.75 in needle x 12 in. tubing Blood Collection Set</td><td>BD</td><td>367297</td><td>Used for cannulation of subhepatic IVC during liver perfusion</td></tr><tr><td>21 G 1 1/2 in. Needles</td><td>BD</td><td>305156</td><td></td></tr><tr><td>1 mL syringe with rubber stops</td><td>BD</td><td>309659</td><td></td></tr><tr><td>10 mL Syringes</td><td>BD</td><td>309604</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>F4/80 PE</td><td>Biolegend</td><td>123110</td><td></td></tr><tr><td>CD11c PE/Cy7</td><td>Biolegend</td><td>117318</td><td></td></tr><tr><td>CD11b PE/Cy5</td><td>eBioscience</td><td>15-0112-81</td><td></td></tr><tr><td>Anti-mouse CD16/32 Fc Block&amp;nbsp;</td><td>Biolegend</td><td>101320</td><td></td></tr><tr><td>CD45 Pacific Blue</td><td>Biolegend</td><td>103126</td><td></td></tr><tr><td>PE Rat IgG2a</td><td>Biolegend</td><td>400508</td><td></td></tr><tr><td>PE/Cy7 Armenian Hamster IgG</td><td>Biolegend</td><td>400922</td><td></td></tr><tr><td>PE/Cy5 Rat IgG2b</td><td>Biolegend</td><td>400610</td><td></td></tr><tr><td>Pacific Blue Rat IgG2c</td><td>Biolegend</td><td>400717</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)&amp;nbsp;</td><td>Cellgro</td><td>15-017-CV</td><td></td></tr><tr><td>1x Phosphate Buffered Saline (PBS)</td><td>Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>70 &amp;mu;m cell strainers</td><td>Corning, Inc.</td><td>352350</td><td></td></tr><tr><td>1.7 mL microcentrifuge tube&amp;nbsp;</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Paraformaldehyde Aqueous Solution -16x</td><td>Electron Microscopy Sciences</td><td>CAS #30525-89-4</td><td></td></tr><tr><td>Micro Dissecting Scissors, 3.5 inch, Straight, 23 mm, Sharp&amp;nbsp;</td><td>Stoelting</td><td>52132-10P</td><td>Used for general dissecting purposes</td></tr><tr><td>Micro Forceps, 4 inches, full curve, 0.8 mm</td><td>Stoelting</td><td>52102-37P&amp;nbsp;</td><td>Used for general dissecting purposes</td></tr><tr><td>Spring dissection scissors &amp;ndash; 3 mm Cutting edge&amp;nbsp;</td><td>Fine Science Tool</td><td>15000-10</td><td>Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td></tr><tr><td>Curved 0.07 mm x 0.04&amp;nbsp;mm Tip Forceps</td><td>Fine Science Tool</td><td>11297-10</td><td>Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td></tr><tr><td>Hemostatic Forceps (Curved)</td><td>Fine Science Tool</td><td>13021-12</td><td></td></tr><tr><td>Heparin Sodium Salt</td><td>Fischer Scientific</td><td>9041-08-1</td><td></td></tr><tr><td>35 mm Cell Culture/Petri Dishes</td><td>Fischer Scientific</td><td>12-565-90</td><td></td></tr><tr><td>Polystyrene Petri Dishes (10 cm) w/lid</td><td>Fischer Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>15 mL Conical Centrifuge Tubes (Polypropylene)</td><td>Fischer Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>50 mL Conical Centrifuge Tubes&amp;nbsp;(Polypropylene)</td><td>Fischer Scientific</td><td>14-432-22</td><td></td></tr><tr><td>5 mL Round-Bottom Polystyrene Tubes&amp;nbsp;</td><td>Fischer Scientific</td><td>14-959-5</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous)</td><td>Millipore</td><td>EX0285-1</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Rockland</td><td>BSA-50</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Collagenase Type II</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>Collagenasse Type XI</td><td>Sigma-Aldrich</td><td>C7657</td><td></td></tr><tr><td>Hyaluronidase Type I</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>DNAse</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Collagenase Type I</td><td>Sigma-Aldrich</td><td>C0130</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>1310-73-2&amp;nbsp;</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>449709-10G</td><td></td></tr><tr><td>500 mL beaker&amp;nbsp;</td><td>Sigma Aldrich</td><td>02-540M</td><td></td></tr><tr><td>4 cm Hemostatic clamp</td><td>Stoelting</td><td>52120-40</td><td></td></tr><tr><td>Toothed forceps</td><td>Stoelting</td><td>52104-33P</td><td></td></tr><tr><td>50 &amp;mu;m Disposable filters</td><td>Systemex</td><td>04-0042-2317</td><td></td></tr><tr><td>Collagenase Type IV</td><td>ThermoFischer Scientific</td><td>17104019</td><td></td></tr><tr><td>Ammonium Chloride Potassium (ACK)</td><td>ThermoFischer Scientific&amp;nbsp;</td><td>A1049201</td><td></td></tr><tr><td>Razors (0.22 mm (0.009&quot;))</td><td>VWR International</td><td>55411-050</td><td></td></tr><tr><td>Texas Red Live/Dead stain</td><td></td><td></td><td>Red viability stain (in &lt;strong&gt;Figure 1A&lt;/strong&gt;)</td></tr></tbody></table>

isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

When using apolipoprotein E deficient (ApoE KO) C57BL/6 (BL6) mice maintained on a high fat high cholesterol diet (HCHFD or HCD) for 18 weeks, 1 x 104 - 2 x 104 CD45+F4/80+ aortic macrophages can be isolated when two samples are pooled. Livers dissected from HFHCD-fed ApoE KO mice, produced greater than 5 x 105 sorted Kupffer cells (which depends on available sorting time). When using high fat diet (HFD) fed wild type (WT) C57BL/6 mic...

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

The overall goal of this procedure is to effectively isolate and characterize macrophages from tissues commonly affected by diet-induced inflammation such as the liver. This method can help answer key questions in the fields of immunology and inflammation about how tissue-resident macrophage phenotype can dictate the progression of chronic inflammatory diseases. The main advantage of this is that the procedure optimizes the isolation steps needed for obtaining single-cell suspensions derived from tissues impacted by obesity-mediated low-grade chronic inflammation.Demonstrating the procedure will be Joselyn Allen, a graduate student from my laboratory. To isolate the liver, first place an ethanol-soaked mouse in the supine position on a polystyrene foam dissecting board and secure the fore and hind paws. Next, use a medium point tip forceps to grasp the abdominal skin near the urethra opening and use sharp dissecting scissors to create a small incision in the raised skin.Insert the lower blade of the scissors into the incision between the skin and the peritoneum and make a lateral incision from the groin to the chin. Gently pull back the skin to expose the intact peritoneal cavity and thoracic wall and make a lateral incision through the peritoneum. Lift the sternum and carefully incise the diaphragm.Then move the gastrointestinal organs to the side and locate the subhepatic inferior vena cava or IVC. Using hemostatic forceps, clamp the suprahepatic IVC to maintain a localized perfusion and attach a syringe filled with pre-warmed perfusion buffer to a 23 gauge blood collection and infusion set. Gently depress the plunger until the tubing and needle are filled with perfusion buffer and insert the needle parallel to the subhepatic IVC.Proper cannulation of the IVC ensures that the dissociation buffer is locally distributed throughout the liver and peripheral tissues optimizing the tissue digestion. Use a four centimeter hemostatic clamp to secure the needle in place and gently depress the plunger to begin the perfusion. If the needle is appropriately positioned, the color will rapidly flush from the liver and the lobes will become enlarged.Promptly cut the portal vein to allow the perfusion buffer to flow freely through the liver, occasionally gently massaging the lobes between the thumb and forefinger to facilitate the removal of the blood. When only five milliliters of perfusion buffer are left, replace the perfusion buffer syringe with a syringe filled with pre-warmed dissociation buffer, continuing to gently massage the lobes and perfuse the liver until it is fully digested. Confirm a successful digestion by gently pressing the blunt edge of a pair of forceps into the left lateral lobe.An indentation should appear on the surface that slowly fills once the forceps is removed. Then remove the needle and use a short straight blade from a pair of dissecting scissors to carefully cut through the connecting ligaments to excise the whole liver and the gallbladder. Transfer the liver to a 10 centimeter dish containing 10 milliliters of ice cold DMEM and use toothed forceps to grip the severed suprahepatic IVC.Using medium point forceps, apply a rapid stroking motion to each lobe to release the cells from the Glisson's Capsule and pass the saturated DMEM through a 70 micrometer cell strainer in a 50 milliliter conical tube on ice. High yields of viable cells can only be achieved by properly dissociating the liver cells from the Glisson's Capsule. When all of the cells have been collected, invert the tube to gently mix the cell suspension and pellet the cells by centrifugation.Transfer the supernatant to a new 50 milliliter conical tube for three more low-spin centrifugations. After the last centrifugation, transfer the supernatant to a new 50 milliliter tube and centrifuge the cells one more time at a higher speed. Then resuspend the non-parenchymal cell pellet in FACS buffer for counting and dilute the liver cells to the appropriate concentration according to their downstream application in labeled five milliliter round bottom polystyrene tubes.Mice fed a high fat diet or a high fat high cholesterol diet exhibit an increased infiltration of classically activated M1 macrophages in affected tissues such as the aorta. RON receptor expressing aortic CD45 positive F4/80 positive macrophages which demonstrate an anti-inflammatory phenotype are decreased in high fat high cholesterol diet fed mice while pro-inflammatory CD45 positive F4/80 positive CD11c positive macrophages are found in increased numbers in aortas isolated from these animals. Further, sorted RON receptor expressing macrophages derived from digested aortas display an increased expression of Arginase 1 gene, a well-established M2 macrophage marker.Indeed, the characterization of liver-resident macrophages reveals the prevailing phenotype of RON receptor expressing subpopulations with CD11c positive pro-inflammatory macrophage populations demonstrating a decreased expression of genes that are strongly associated with the ant-inflammatory M2 phenotype. Similar trends are observed in the macrophage populations isolated from digested white adipose tissue with pro-inflammatory phenotype macrophage populations exhibiting a decreased surface expression of the RON receptor. Once mastered, this technique can be completed in 20 to 30 minutes per animal if it is performed properly.Following this procedure, other methods such as flow cytometry, fluorescent-activated cell sorting, and/or quantitative PCR can be performed to further characterize the phenotype of the isolated liver-derived tissue-resident macrophages. After watching this video, you should have a good understanding of how to properly isolate tissue-resident macrophages from diseased livers. Thanks for watching and good luck with your experiments.

isolation-characterization-purification-macrophages-from-tissues

Research

JoVE Journal

Immunology and Infection

A novel method to obtain macrophages from primary culture of rat liver cells is described.  This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Adipose tissue (AT) is a site of intense immune cell activation and interaction.  Almost all cells of the immune system are present in AT and their ratios are altered by obesity.  Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Isolation of Adipose Tissue Immune Cells

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteomic Profiling of Macrophages by 2D Electrophoresis

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Learn to isolate and characterize macrophages from liver tissue affected by obesity-related inflammation. This optimized protocol enables efficient single-cell suspension for immunology and metabolic disorder research.

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

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