Isolation of Adipose Tissue Immune Cells

Summary

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.

Abstract

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.

Introduction

Historically, the adipose tissue (AT) has been viewed as an inert organ of lipid storage, which expands and contracts in response to energy balance. We now understand that AT represents a dynamic endocrine organ that actively secretes a number of hormones, which directly influence feeding behavior and systemic glucose homeostasis. In addition, over the past decade there has been an increasing appreciation for the numerous populations of immune cells residing in the AT stromal vascular fraction (SVF), as well as their contribution to AT homeostasis.

The ability to separate the AT adipocyte and SVF using a collagenase digest fol....

Protocol

1. Reagents and Supplies

Prior to initiating this experimental protocol, prepare the following reagents:

1. 70% ethanol
2. 1X PBS
3. 1X DPBS (without Ca and Mg) supplemented with 0.5% BSA
4. FACS buffer: 1X DPBS (without Ca and Mg), 2 mM EDTA, and 1% FCS
5. ACK buffer: 150 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM Na₂EDTA in water

2. Harvesting and Preparation of Adipose Tissue

1. Euthanize mice according to IACUC-approved procedures specific to each institution.
2. Thoroughly wet the fur with 70% ethanol.
3. Make an incis....

Results

Collagenase digestion of AT followed by differential centrifugation was used to isolate the SVF from epididymal fat pads of male C57BL/6J mice fed a low fat (10% kcal from fat) or high fat (60% kcal from fat) diet (LFD and HFD, respectively) for 16 weeks. Cells of the SVF were then labeled with fluorophore-conjugated primary antibodies to quantify the proportion of viable ATMs (Figure 1) and AT T cells (Figure 2) via FACS analysis. Initial gating, including light scatter, doublet .......

Discussion

Increasing interest in the role of the immune system in the metabolic consequences of obesity has led to the widespread use of flow cytometry to characterize immune cells of the AT. Although the exact protocol will vary between laboratories based on their own experience and available equipment, the critical steps include collagenase digestion, differential centrifugation, and cell surface antigen labeling. The goal of the present article is to provide a detailed protocol and practical guide for the isolation of the AT SV.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DPBS (no Ca or Mg) 10 x 500 ml	Life Technologies	14190-250
DAPI	Life Technologies	D3571
BSA	Sigma	A2153-100G
collagenase	Sigma	C6885-5G
propidium iodide solution	Sigma	P4864-10 ml
stable stack 20 microliter	Rainin	SS-L10
20 microliter filter tips	Rainin	SRL10F
stable stack 250 microliter tips	Rainin	SS-L250
1000 microliter tips	Rainin	GPS-L1000
1000 microliter filter tips	Rainin	GP-L1000F
250 microliter Filter Tips	Rainin	SR-L200F
FC Block	BD Biosciences	553142
fisher 100mn strainers	Fisherbrand	22-363-549
medium weigh dish	Fisherbrand	02-202B
aluminum foil	Fisherbrand	1213100
mincing scissors	Fisherbrand	089531B
Vortex	Fisherbrand	2215365
50 ml conical tubes	BD Falcon	14-959-49A
filter top FACS tubes	BD Falcon	352235
10 ml pipette case 200	BD Falcon	1367520
round bottom tubes	BD Falcon	352058
5 ml syringe	BD Falcon	309646
V Bottom Plates	Costar	07-200-107
transfer bulb pipette	Thermo Scientific	13-711-22
Shaker	Thermo Scientific	11 676 071
Adhesive mat	Thermo Scientific	1368750
Cell Culture Centrifuge	Sorvall	75253839
Adapters	Sorvall	75003723
Rat anti-mouse CD16/CD32	BD Biosciences	553142	Concentration: 0.5 - 1 μg/ 106 cells
Rat anti-mouse F4/80	eBioscience	17-4801	Fluorophore conjugate: APC Concentration: 0.2 μg/ 106 cells Isotype control catalog number: 17-4321
Rat anti-mouse CD11b	eBioscience	11-0112	Fluorophore conjugate: FITC Concentration: 0.5 μg/ 106 cells Isotype control catalog number: 11/1/4031
Armenian Hamster anti-mouse CD11c	eBioscience	12-0114	Fluorophore conjugate: PE Concentration: 0.8 μg/ 106 cells Isotype control catalog number: Dec-88
Goat anti-mouse MGL1/2	R&D Systems	FAB4297P	Fluorophore conjugate: PE Concentration: 0.1 μg/ 106 cells Isotype control catalog number: IC108P
Goat anti-mouse CD206	R&D Systems	FAB2535P	Fluorophore conjugate: PE Concentration: 0.1 μg/ 106 cells Isotype control catalog number: IC108P
Rat anti-mouse CCR2	R&D Systems	FAB5538P	Fluorophore conjugate: PE Concentration: 0.1 μg/ 106 cells Isotype control catalog number: IC013P
Armenian Hamster anti-mouse TCRβ	BD Biosciences	553174	Fluorophore conjugate: APC Concentration: 0.2 μg/ 106 cells Isotype control catalog number: 553956
Rat anti-mouse CD8a	BD Biosciences	552877	Fluorophore conjugate: PE-Cy7 Concentration: 0.8 μg/ 106 cells Isotype control catalog number: 552784
Rat anti-mouse CD4	BD Biosciences	557956	Fluorophore conjugate: Alexa Fluor 700 Concentration: 0.2 μg/ 106 cells Isotype control catalog number: 557963
Table 1. List of Materials and Reagents.