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

Summary

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.

Abstract

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.

Introduction

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 disease^2,3. Excessive nutrient consumption is further skewed by decreased physical activity triggering alter....

Protocol

All experimental protocols (Sections 1, 1.2, and 1.3) were approved by the Institutional Animal Care and Use Committee (IACUC) at Pennsylvania State University.

Tissue	Dissociation Buffers Preparation	Final Volume	Storage
White Adipose Tissue (WAT)	Dissociation Buffer: 2.5% HEPES, 10 mg/mL Bovine serum albumin (BSA), 3 mg/mL (0.3%) Collagenase Type II in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose without L-glu....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
26 G x 5/8 in Needles	BD	305115
23 G x 0.75 in needle x 12 in. tubing Blood Collection Set	BD	367297	Used for cannulation of subhepatic IVC during liver perfusion
21 G 1 1/2 in. Needles	BD	305156
1 mL syringe with rubber stops	BD	309659
10 mL Syringes	BD	309604
1 mL Syringe	BD	309659
F4/80 PE	Biolegend	123110
CD11c PE/Cy7	Biolegend	117318
CD11b PE/Cy5	eBioscience	15-0112-81
Anti-mouse CD16/32 Fc Block	Biolegend	101320
CD45 Pacific Blue	Biolegend	103126
PE Rat IgG2a	Biolegend	400508
PE/Cy7 Armenian Hamster IgG	Biolegend	400922
PE/Cy5 Rat IgG2b	Biolegend	400610
Pacific Blue Rat IgG2c	Biolegend	400717
Dulbecco’s Modified Eagle Medium (DMEM)	Cellgro	15-017-CV
1x Phosphate Buffered Saline (PBS)	Cellgro	21-031-CV
70 μm cell strainers	Corning, Inc.	352350
1.7 mL microcentrifuge tube	Denville	C2170
Paraformaldehyde Aqueous Solution -16x	Electron Microscopy Sciences	CAS #30525-89-4
Micro Dissecting Scissors, 3.5 inch, Straight, 23 mm, Sharp	Stoelting	52132-10P	Used for general dissecting purposes
Micro Forceps, 4 inches, full curve, 0.8 mm	Stoelting	52102-37P	Used for general dissecting purposes
Spring dissection scissors – 3 mm Cutting edge	Fine Science Tool	15000-10	Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28
Curved 0.07 mm x 0.04 mm Tip Forceps	Fine Science Tool	11297-10	Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28
Hemostatic Forceps (Curved)	Fine Science Tool	13021-12
Heparin Sodium Salt	Fischer Scientific	9041-08-1
35 mm Cell Culture/Petri Dishes	Fischer Scientific	12-565-90
Polystyrene Petri Dishes (10 cm) w/lid	Fischer Scientific	08-757-100D
15 mL Conical Centrifuge Tubes (Polypropylene)	Fischer Scientific	14-959-53A
50 mL Conical Centrifuge Tubes (Polypropylene)	Fischer Scientific	14-432-22
5 mL Round-Bottom Polystyrene Tubes	Fischer Scientific	14-959-5
Fetal Bovine Serum	Gemini Bio-Products	100-106
Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous)	Millipore	EX0285-1
Bovine Serum Albumin	Rockland	BSA-50
HEPES	Sigma-Aldrich	H3375
Collagenase Type II	Sigma-Aldrich	C6885
Collagenasse Type XI	Sigma-Aldrich	C7657
Hyaluronidase Type I	Sigma-Aldrich	H3506
DNAse	Sigma-Aldrich	DN25
Collagenase Type I	Sigma-Aldrich	C0130
NaOH	Sigma Aldrich	1310-73-2
CaCl2	Sigma Aldrich	449709-10G
500 mL beaker	Sigma Aldrich	02-540M
4 cm Hemostatic clamp	Stoelting	52120-40
Toothed forceps	Stoelting	52104-33P
50 μm Disposable filters	Systemex	04-0042-2317
Collagenase Type IV	ThermoFischer Scientific	17104019
Ammonium Chloride Potassium (ACK)	ThermoFischer Scientific	A1049201
Razors (0.22 mm (0.009"))	VWR International	55411-050
Texas Red Live/Dead stain			Red viability stain (in Figure 1A)