Name: isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation
Uploaded: 2017-04-03T14:00:00.000+00:00
Duration: 7 min 46 s
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

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

<ol>
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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 neighboring cells. The tissues&#39; structural composition dictates the tissue matrix stiffness (resistance to deformation) and which crude collagenase product is most efficient in ensuring successful ECM disruption28. WAT, which is composed of &#34;soft matrix&#34; is often digested with Collagenase Type II to liberate adipocytes and resident immune cells while simultaneously maintaining the integrity of cell surface insulin receptors29. The microarchitecture of the fibril components of the aorta contributes to the &#34;stiff matrix&#34;, for which effective aortic digestion with collagenase type XI (which has the highest collagenase activity) is used in combination with additional enzymes. Unlike the aorta, the liver digestion uses a collagenase with a weaker enzymatic activity, Collagenase Type IV, to disrupt the matrix and liberate viable parenchymal and non-parenchymal cells30. Chronically inflamed tissues derived from diet-fed wild type or apolipoprotein E deficient (ApoE KO) mice on a C57BL6 background often undergo remodeling that can prevent proper enzymatic digestion. This section will discuss strategies to minimize and/or eliminate the possibility of improper tissue dissociation and low cell recovery.
A common feature of tissues derived from diet-fed mouse models that mimic obesity, atherosclerosis, and/or non-alcoholic fatty liver disease, is abnormal tissue remodeling. In white adipose tissue, the aorta, and liver, ECM composition is altered, often resulting in excessive deposition of fibrillar components such as collagens. Wild type C57BL6 mice maintained on a high fat diet for eighteen weeks, experience tissue-specific abnormalities such as expanded white adipose tissue and enlarged fatty liver (simple steatosis). Often times these metabolic features are not troublesome obstacles to overcome during the tissue dissociation process. On the other hand, in other diet induced models that mirror more exacerbated phenotypes, the extensive remodeling of the tissue can pose a problem during tissue digestion. HFHCD fed ApoE KO mice are often used to model atherosclerosis and steatohepatitis. A common feature associated with advanced steatohepatitis is the excessive deposition of ECM in the liver (or fibrosis). Fibrotic liver has been shown to be quite problematic during enzymatic tissue dissociation and often produces low cell yield31. To improve cell yield, it is critical that the digestion protocols be followed without deviation. Although normal livers can be minced and submerged in digestion buffer to achieve dissociation, perfusion with digestion buffer maximizes contact between the extracellular matrix of the liver and the collagenase solution; and therefore this approach is highly recommended. Additionally, 20-30 mL of digestion buffer is often an adequate volume for successful dissociation of normal livers; however, in regards to mouse models that develop enlarged and/or fibrotic livers, perfusion with 50 mL of digestion buffer is preferred to ensure proper digestion while minimizing cell death. For livers with weights that significantly exceed 1.5 g, increasing the volume of digestion buffer is recommended to guarantee successful digestion.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tissue&#160;</td>
			<td>Problem</td>
			<td>Possible Reason&#160;</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>White Adipose Tissue (WAT)</td>
			<td>Poor cell dissociation&#160;</td>
			<td>Poor Digestion</td>
			<td>Make sure digestion buffer is at 37 &#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td>Cell Death</td>
			<td>Excessive collagenase digestion</td>
			<td>Reduce time of digestion</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reduce volume of digestion buffer</td>
		</tr>
		<tr>
			<td>Aorta</td>
			<td>Poor cell dissociation&#160;</td>
			<td>Poor Digestion</td>
			<td>Make sure digestion buffer is at 37 &#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Aorta pieces in digestion buffer were too large</td>
			<td>Make sure aorta is cut into approximately 1 mm pieces</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Aorta pieces were not shaking in dissociation buffer during incubation</td>
			<td>Be sure aorta pieces are shaking in the digestion solution</td>
		</tr>
		<tr>
			<td></td>
			<td>Cell Death</td>
			<td>Excessive collagenase digestion</td>
			<td>Reduce time of digestion</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reduce volume of digestion buffer</td>
		</tr>
		<tr>
			<td>Liver</td>
			<td>Poor cell dissociation&#160;</td>
			<td>Poor Digestion</td>
			<td>Make sure digestion buffer is at 37 &#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Increase volume of digestion buffer</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Improper cannulation of IVC (swelling of tissue surrounding IVC occurs)</td>
			<td>Be sure needle is properly inserted into the IVC</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Ruptured IVC</td>
			<td>Properly secure needle in IVC prior to perfusion</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reduce perfusion speed</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Ruptured tissue</td>
			<td>Reduce perfusion speed</td>
		</tr>
		<tr>
			<td></td>
			<td>Cell Death</td>
			<td>Improper release of cells from the Glisson&#8217;s capsule</td>
			<td>Be sure to dissociate cells from capsule using previously discussed stroking method</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Excessive collagenase digestion</td>
			<td>Reduce time of digestion</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reduce volume of digestion buffer</td>
		</tr>
	</tbody>
</table>
Table 3: Troubleshooting unsuccessful tissue dissociation. Flow cytometry based cell sorting or FACS is a powerful technique for isolating cell populations where high purity is a necessity. When purifying cells by cell sorting, achieving high cell yield but also a high purity sort requires using the proper sorting strategies. In this section, methods for improving multi-fluorophore flow cytometry-based cell sorting of tissue resident macrophage derived from diet-fed mice are described. For distinct tissue resident macrophage isolation, surface marker selection is a critical step. Macrophages derived from WAT, aorta, and liver are often distinguished using a CD45+, CD11b+, F4/80+ gating strategy. Additional flow cytometric panels can be used to identify tissue resident macrophages. These panels include antibodies which probe for the surface expression of macrophage specific glycoproteins (CD64, CD68, and CD14), major histocompatibility complexes (MHCII) and apoptotic cell tyrosine kinase receptors (MerTK)32,33. Specific macrophage phenotype can then be delineated by probing for the selective surface expression of M2 (CD163, CD209, and CD206) or M1 markers (CD38, CD40, CD80, and CD86)34,35. Auto-fluorescence generated by lipid-laden macrophages can present some issues when gating populations. Using antibodies tagged with fluorochromes that are excited by the yellow-green laser (such as PE, PE/Cy5, PE/Cy7) or red laser (such as APC, APC Cy7) results in emitted fluorescence that is significantly brighter than the auto-fluorescence and thus can improve results36. When selecting fluorophore conjugates with such high staining index and potential emission spectra overlap, inclusion of appropriate controls is critical. When identifying gating boundaries, inclusion of single stain (SS) controls and isotype controls allow for the delineation of positive/negative populations and the measurement of non-specific background signal caused by primary antibodies, respectively37. For circumstances where cells are scarce, we recommend using compensation particles for SS and isotype controls. Compensation particles typically emit brighter signals than biological controls and also have less variance in background fluorescence. Additionally, fluorescence minus one (FMO) controls are ideal for delineating gating boundaries. By including FMO controls, the maximum fluorescence expected for a staining subset is revealed in a given channel when the fluorochrome-tagged antibody specific for that particular fluorescence channel is excluded38. In our experience, in addition to single stained compensation particle controls, an unstained biological comparison control should be included for setting more precise positive/negative boundaries.
Excluding cellular debris and cell aggregates in the gating strategy is also an additional approach used to minimize auto-fluorescence. To distinguish cellular debris from the viable cell population, using forward scatter (FSC) and side scatter (SSC) is the most common gating strategy. For isolated cells that will be used for post sort analyses and require higher viability for DNA/RNA extractions, it is recommended that viability stains not be used with any fixation and/or permeabilization procedures. Formaldehyde fixing of cells can compromise nucleic acid integrity due to nucleic acid-protein crosslinking and thus limit isolation efficiency, detection, and accurate quantification. Cell aggregates as mentioned before cannot only contribute to emitted auto-fluorescent signals, but also result in &#34;coincidence aborts&#34;. Such action occurs to maintain high purity in a sort but if too frequent, it reduces the sorted cell yield. Sorting buffer (FACS buffer) supplemented with DNAse and MgCl2 during cell staining can minimize cell aggregation. It is recommended to not use EDTA in combination with DNAse as it inhibits the enzyme&#39;s activity. Filtering the single cell suspension through a 70 &#181;m cell strainer prior to sorting can also liberate cells from aggregates. It is imperative to note that to minimize aggregation, cell suspensions should be at a concentration of 5 million cells per mL in a minimum volume of 0.4 mL. Cells isolated from lipid laden tissues tend to be more prone to aggregation and this can result in coincidence aborts and low sort yield. It is recommended that samples are further diluted if aggregation persists. Sorted macrophages can be collected in polypropylene round-bottom collection tubes containing fetal bovine rich medium to maximize cell recovery. An initial high FBS concentration ensures cell recovery since the concentration eventually becomes diluted with each sorted droplet. For collecting cells that will be used for DNA or RNA analysis, cells can be sorted directly into the appropriate extraction reagent (e.g. TriZol) to prevent RNAse contamination. When sorting high volumes, sorting cells first into culture media supplemented with lower concentrations of FBS, is recommended. Immediately following sorting, cells should then be pelleted and lysed for DNA/RNA extraction. The isolated genetic material can then be profiled for altered gene expression. Pro-inflammatory genes including TNF&#945;, IL1-&#946;, IL-6, IL-12, 1L-23, IFN&#947;, Nos2, and MCP1 (CCL2) are often upregulated in macrophages exhibiting a classically activated (M1) phenotype39. On the other hand, alternatively activated (M2) phenotype in macrophages is often marked by induction of genes encoding Chi3l3 (Ym1), Fizz1, Arginase 1, CD206, CD163, CD209 1L-10, and TGF&#946;34,40. Recently, CD38, Fpr2, and Gpr18 have been validated as M1-specific genes and c-Myc and Egr2 as M2-specific genes34.
Although multi-color flow cytometry-based cell sorting is a valuable tool for isolating macrophages at high purity, this approach can be costly. The powerful advantages of FACS mediated sorting are dependent on operations personnel that can maneuver a cell sorter in addition to the high cost of cell sorter maintenance reagents. Alternative approaches can be used in substitute of costly flow cytometry based cell sorting. They include magnetic activated cell sorting (MACS) or density gradient centrifugation. The first alternative cell sorting method mentioned encompasses magnetic and/or microbead column isolation kits to separate cells of interest from blood or solid tissues41. The second cell sorting approach separates a heterogeneous cell suspension based on density and force of centrifugation. Unfortunately, density mediated centrifugation is not practical for isolating macrophages from aorta- or WAT-derived single cell suspensions. Oftentimes, the product obtained from differential centrifugation is contaminated, and of low yield. Consequently, smaller tissues (such as the aorta or WAT) that result in few cells initially following enzymatic digestion are not ideal candidates for differential centrifugation. On the other hand, cell suspensions derived from dissociated livers can produce an adequate number of sorted macrophages that can be used in post sort experiments and analyses such as culture stimulations, qPCR, or western blotting analysis. These alternative approaches can also be used to enrich populations prior to FACS, allowing for cleaner sorts. Of note, tissue resident macrophages make up a small percentage of the entire cell population in WAT, liver and the aorta. Enrichment of cells prior to FACS sorting has been an approach used when isolating populations of cell that are less frequent. One issue that is common in isolating small populations is that large cell numbers must be processed to obtain enough cells for subsequent analysis. Enrichment or pre-sorting can be used to resolve such an issue. This method aids in obtaining a more concise population of cells through positive and negative selection but it also allows conservation of time as FACS sorting can be an enduring process for dense tissue sources such as the liver.
Recent advances in inflammation biology highlight the importance of phenotypic and functional characterization of macrophage heterogeneity to further the understanding of the complex role of these immune cells in regulating chronic inflammation. In brief, this comprehensive protocol provides a multi-dimensional approach to characterizing tissue resident macrophages from three hallmark tissues studied in established diet induced obesity and inflammation models. More importantly this protocol takes into account the difficulty and the measures necessary to isolate clean single cell suspensions from dysregulated inflamed tissues such as WAT, aortic plaques and fatty livers. The protocol allows the researcher to apply flow cytometry and FACS sorting tools in an innovative dimension to characterize tissue resident macrophages, key regulators of inflammation in obesity. In-depth characterization of macrophage population dynamics can provide insight into monocyte trafficking in inflamed tissues and allow for continued mechanistic evaluations through a multitude of experimental approaches on sorted macrophages. Further characterization of macrophage populations can provide a pivotal insight into the biological underpinnings that regulate macrophage heterogeneity in health and disease.

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

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 mice, 5 x 105 to 1 x 106 resident adipose tissue macrophages (ATMs) can be sorted from the stromal vascular fraction (SVF). The mentioned total number of macrophages that can be sorted from a given tissue was adequate for performing gene expression analysis using quantitative real time polymerase chain reaction (qPCR). For tissues where fewer numbers of macrophage populations are recovered, such as the aorta, it is recommended to use co-precipitants (for example glycogen) and overnight precipitation when isolating RNA is needed for these analyses.
Here, the effects of diet-induced metabolic disorders on macrophage phenotype using basic flow cytometry, fluorescence-activated cell sorting (FACS), and downstream post sort analyses are shown. These findings corroborate previously published observations that mice fed a HFD or HFHCD exhibit increased infiltration of classically activated (M1) macrophages in affected tissues such as the aorta (Figure 1B). Taking advantage of flow cytometry, FACS, and gene expression profiling (via quantitative polymerase chain reaction (qPCR), the predominating phenotype for Ron receptor tyrosine kinase-expressing CD45+ F4/80+ macrophages derived from tissues isolated from diet-fed mice was observed. Ron receptor-expressing aortic macrophages which demonstrated an anti-inflammatory phenotype were decreased in HFHCD-fed mice (Figure 1C and D). Sorted Ron receptor-expressing macrophages derived from digested aortas demonstrated increased arginase 1 (Arg1) gene expression, which is a well-established M2 macrophage marker (Figure 1E). Pro-inflammatory macrophages which were characterized as CD45+ F4/80+ CD11c+ were increased in aortas isolated from HFHCD-fed mice (Figure 1B and D). Characterizing liver-resident macrophages further elucidated the prevailing phenotype of Ron receptor-expressing subpopulations (Figure 2A). CD11c+ pro-inflammatory macrophage populations demonstrated a decrease expression of genes that are strongly associated with an anti-inflammatory (M2) phenotype such as Arg1 and Ron (Figure 2B). Similar trends were observed in macrophage populations isolated from digested white adipose tissue (Figure 3). Macrophage population with a pro-inflammatory signature showed decreased surface expression of the Ron receptor (Figure 3). Combining the approaches, basic flow cytometry and FACS, resulted in conclusive data that further validates the Ron receptor as a regulator of alternative (M2) activation in macrophages32. Such bias toward an anti-inflammatory (M2) phenotype has been shown to play a protective role in the development and progression of obesity, atherosclerosis and steatohepatitis31.
<img alt="Figure 1" src="/files/ftp_upload/55445/55445fig1.jpg" /> 
Figure 1: Characterization of macrophages isolated from dissociated aorta removed from ApoE KO mice maintained on a HCD for 18 weeks. (A) Cells were first gated on CD45+ leukocytes, excluding cellular debris. (B) CD45 staining in conjunction with F4/80 is used to delineate double positive populations presumed to be macrophages. Additional gating was used to demonstrate the percentage of CD11c+CD45+F4/80+ (M1) and (C) Ron+CD45+F4/80+ (potential M2) macrophages in aortas derived from mice fed on either a normal chow or high cholesterol diet for 18 weeks. (D) Increased percentage of CD11c+CD45+F4/80+ (M1) macrophages, as well as a decreased percentage of Ron+CD45+F4/80+ (potential M2) macrophages were observed in aortas derived from mice fed HCD compared to mice fed a normal chow diet. (E) Gene expression analysis of sorted Ron+CD45+F4/80+ macrophages demonstrated increased expression of arginase I (a well-known murine M2 marker). Values were obtained using Student&#39;s t-test analyses performed using statistical analysis software and represented as mean &#177; SEM. P &#60; 0.05 was considered statistically significant (*P &#60; 0.05, ***P &#60; 0.001). Figure has been modified from Yu et al. (2016)31. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55445/55445fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55445/55445fig2.jpg" /> 
Figure 2: Gene transcript profiling of Kupffer cell populations sorted from digested livers dissected from ApoE KO mice maintained on a HCD for 18 weeks. (A) General gating scheme for characterizing and sorting Kupffer cell populations. (B) Gene expression analysis of sorted Ron expressing (Ron+) and non-expressing (Ron-) CD45+F4/80+ macrophages by quantitative RT PCR. Student&#39;s t-test analyses were performed using statistical software and values were represented as mean &#177; SEM. P &#60; 0.05 was considered statistically significant (*P &#60; 0.05, ***P &#60; 0.001). Figure has been modified from Yu et al. (2016)31. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55445/55445fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55445/55445fig3.jpg" /> 
Figure 3: Characterization of ATMs isolated from white adipose tissue dissected from WT BL6 mice fed a HFD for 18 weeks. (A) General gating scheme for characterizing and sorting adipose tissue derived macrophage populations (B) Percentage of Ron+ cells within CD45+F4/80+CD11c- and CD45+F4/80+CD11c+ macrophage populations sorted from WAT. Figure has been modified from Yu et al. (2016)31. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55445/55445fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

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

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. ...

isolation-characterization-purification-macrophages-from-tissues

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

25.1K Views.  The Pennsylvania State University. Dit protocol kan een onderzoeker te isoleren en karakteriseren weefsel-ingezeten macrofagen in diverse hallmark ontstoken weefsels dieet-geïnduceerde modellen van metabolische wanorde is onttrokken.

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

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

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

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.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

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.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Biochemistry

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

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.

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.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

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.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

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

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

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.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

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

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

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.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Neuroscience

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

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

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.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Medicine

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

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

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

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.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

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

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