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 followed by differential centrifugation was first described by Rodbell in 1964 ¹. Collagenase II is most often used for adipocyte and SVF separation due to maintenance of adipocyte insulin receptors¹. Early on, enzymatic fractionation of AT was primarily employed to study adipocyte metabolism and to isolate preadipocytes. More recently, this technique, combined with the widespread availability of flow cytometers and the ever-increasing number of commercially available fluorophore-conjugated antibodies, has facilitated the characterization of AT immune cells.

Although the presence of immune cells in inflamed AT had been described previously ², the seminal papers by Weisberg et al. and Xu et al. published in 2003 were the first to document the accumulation of AT macrophages (ATMs) in obesity, which secrete inflammatory cytokines and correlate with AT-specific and systemic insulin resistance ^3,4. These observations served as the basis of a new field of investigation recently coined, "immunometabolism," ⁵ and have been followed up by studies implicating various immune cell populations, including dendritic cells ⁶, mast cells ⁷, T cells ^8-10, B cells ¹¹, NKT cells ¹², eosinophils ¹³, and neutrophils ^14,15 in the development of obesity associated insulin resistance.

The goal of this article is to provide a detailed description of the collagenase digest technique used to isolate cells of the AT SVF and to characterize ATMs and AT T cells via flow cytometry. This protocol has been optimized for mouse AT; however, viewers may benefit from reading an excellent article providing extensive detail on optimization of this technique for human AT ¹⁶. The target audience of this article includes investigators with limited experience working with mouse AT and performing flow cytometry. Several practical considerations for balancing cellular yield and viability with time and resources are presented as well as optimal flow cytometry controls for characterizing AT immune cell populations. In addition to our protocol, readers are referred to a recent JoVE article by Basu et al. for an excellent discussion of some of the technical aspects of flow cytometry to include proper controls and compensations ¹⁷.

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 incision at the level of the xiphoid process (lower part of the sternum) and open the thoracic cavity to expose the heart, taking care not to sever any major blood vessels.

Note: At this point, it is also helpful to leave the diaphragm intact as much as possible and to cut a notch out of the right side of the rib cage to allow blood and perfusate to flow out of the thoracic cavity.

4. Clip the right atrium to allow blood and perfusate to escape the circulatory system.

Note: When dealing with obese mice, excess pericardial AT may need to be removed to permit access to the heart.

5. Grasp the heart with forceps and gently insert a needle into the left ventricle through the apex. Slowly perfuse the mouse with 15 ml sterile PBS.

Note: Reduce the rate of perfusion if the lungs begin to fill and expand.

6. Open the peritoneal cavity and remove the perigonadal fat pads using care to avoid any gonadal tissues.
7. Place fat pads in a weigh boat on ice containing 2 ml 1X DPBS (without Mg or Ca) supplemented with 0.5% BSA, and mince the AT into fine pieces.

Note: Limit the amount of AT per weigh boat to 1.2 g. If the amount of AT exceeds 1.2 g, divide it evenly between two weigh boats.

8. Keep AT samples on ice and prepare 3 ml collagenase digest solution per AT sample consisting of 1X DPBS supplemented with 0.5% BSA, 10 mM CaCl₂, and 4 mg/ml collagenase, type II.

3. Collagenase Digestion

1. Transfer AT to 50 ml conical tubes by pouring the homogenate and rinsing the weigh boat with 1 ml DPBS (0.5% BSA) and 3 ml collagenase II digest solution.
2. Incubate AT homogenate in a rotational shaker (200 rpm) at 37 °C for 20 min.
3. Add 10 ml DPBS (0.5% BSA) to conical tubes and place on ice.
4. Triturate homogenate numerous times using a 10 ml serological pipette, and pass cell suspensions through 100 μm filter in to a new 50 ml conical tube.
5. Centrifuge cell suspension at 500 x g for 10 min at 4 °C.
6. Decant supernatant and resuspend SVF cell pellet in 3 ml ACK buffer to lyse contaminating erythrocytes.
7. Add 12 ml FACS buffer and centrifuge cell suspension at 500 x g for 10 min at 4 °C.
8. Decant supernatant and resuspend SVF cell pellet in FACS buffer.

Note: Use the size of the cell pellet as a guide for how much FACS buffer to resuspend in. If the cell pellet covers the bottom of the 50 ml conical tube, use 0.5-1 ml FACS buffer; otherwise, resuspend in 0.25-0.5 ml.

9. Place samples on ice and prepare 1:10 dilution aliquots of each sample for cell counting by mixing 40 μl FACS buffer, 50 μl trypan blue solution (0.2%), and 10 μl cell suspension.
10. Count viable cells based on trypan blue exclusion and dilute cell suspensions to a final concentration of 5-10 x 10⁶ cells/ml.

4. Staining of Cell Surface Antigens

1. Add anti-mouse CD16/CD32 antibody (Fc block) to a final concentration of 0.5-1 μg/10⁶ cells, and incubate on ice for 10 min.
2. Transfer samples (≥10⁶ cells) to 12 x 75 mm polystyrene round bottom tubes. Prepare separate tubes if analyzing ATMs and T cells, and combine extra cells to prepare an adequate number of tubes to accommodate the required compensation and modified fluorescence minus one (FMO) controls.

Note: As an example, when quantifying the proportion of ATMs based on F4/80 and CD11b, the following compensation and FMO controls will need to be prepared:

- Unstained (cells)

- DAPI or propidium iodide (PI) single stain (cells; do not add viability dye until step 5.1)

- F4/80 APC single stain (cells or compensation beads)

- CD11b FITC single stain (cells or compensation beads)

- FMO 1 (cells): Rat IgG2a κ isotype control APC + CD11b FITC + viability dye (added at step 5.1)

- FMO 2 (cells): F4/80 APC + Rat IgG2b κ isotype control FITC + viability dye (added at step 5.1)

3. Add fluorophore-conjugated primary antibodies and/or isotype controls at the appropriate concentration (see Table of reagents and materials).
4. Protect samples from light and incubate at 4 °C for 30 min.
5. Add 2 ml FACS buffer and centrifuge cell suspension at 500 x g for 5 min at 4 °C.
6. Decant supernatant and resuspend SVF cell pellet in 2 ml FACS buffer.
7. Centrifuge cell suspension 500 x g for 5 min at 4 °C, and resuspend SVF cell pellet in ≥400 μl FACS buffer.
8. Transfer samples to 12 x 75 mm polystyrene round bottom tubes equipped with a 35 μm cell strainer tube tops.
9. Protect from light, and store samples at 4 °C until FACS analysis.

Note: For optimum results cells should be analyzed immeadiately; however, FACS analysis with this protocol has been successfully conducted on labeled cells stored in FACS buffer for 1-2 hr at 4 °C. If cells need to be fixed to increase storage time, labeled cells can be fixed with 2% paraformaldehyde at 4 °C for 24 hr prior to FACS analysis. Antibody companies suggest that labeled cells can be stored for up to one week; however, this has not been tested within the context of this procedure.

5. FACS Analysis

1. Prior to FACS analysis, add viability dye to samples and appropriate controls to allow for live/dead cell discrimination.

Note: Numerous viability dyes are commercially available, but DAPI and PI are recommended depending on the excitation/emission profiles of the fluorophore-conjugated antibodies being used. DAPI and Propidium iodide are added to each sample at a final concentration of 0.2 mg/ml.

2. Use an unstained negative control sample, preferably cells isolated from the same tissue as the experimental samples, to adjust side scatter (SSC) and forward scatter (FSC) so that the cell population(s) of interest are on scale.

Note: For the analysis of AT SVF cells, it is recommended that SSC be displayed in a log scale versus FSC in a linear scale. The use of a log scale for SSC is especially important when analyzing ATMs, which are often very large and granular.

3. Draw an initial light scatter gate based on the type of cell(s) being analyzed, and adjust the photomultiplier tube (PMT) gain so that the unstained cells are on the far left of a single-parameter histogram (approximately centered on 10²) for the appropriate channels.

Note: It is recommended that lymphocytes and macrophages (or other myeloid cells) be analyzed separately due to differences in autofluorescence.

4. Use single stained controls or antibody capture compensation beads to perform multi-color compensation.

Note: The use of compensation beads is recommended; however, compatibility of each antibody must be ensured. For example, compensation beads may not cross-react with rabbit antibodies, in which case isolated cells are required to obtain an appropriate single stain control.

5. Set experimental gates based on modified FMO controls.
6. Set the flow cytometer to collect the appropriate number of events based on the prevalence of the population of interest and record experimental data.
7. Export FCS data files for offline analysis. There are numerous programs available for flow cytometry data analysis. We recommend Cytobank, a web-based platform that allows investigatores to store and analyze data and generate figures from any computer with internet access ¹⁸. Additionally, Cytobank offers the ability to make data public accessible or restrict access to collaborators.

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.