Protocol for Seahorse analysis of ex vivo mouse brown and white adipose tissues

Summary

The mitochondrial stress test is a gold-standard approach for assessing adipose tissue physiological functions and pathological changes. Here, we present a protocol for conducting Seahorse assays using ex vivo mouse brown and white adipose depots. We describe steps for rehydrating the cartridge, preparing freshly harvested fat depots, placing them onto an islet capture plate, and incubating them in a non-CO₂ incubator. We then detail procedures for adding mitochondrial stressor solutions and conducting the mitochondrial stress test using the Seahorse XFe24 Analyzer.

For complete details on the use and execution of this protocol, please refer to An et al.¹

Graphical abstract

Highlights

• •Steps for preparing mouse brown and white fat pads for ex vivo Seahorse assays
• •Instructions on collecting fat pads from similar locations and with similar sizes
• •Optimized settings for mitochondrial stress test using ex vivo adipose tissues

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The mitochondrial stress test is a gold-standard approach for assessing adipose tissue physiological functions and pathological changes. Here, we present a protocol for conducting Seahorse assays using ex vivo mouse brown and white adipose depots. We describe steps for rehydrating the cartridge, preparing freshly harvested fat depots, placing them onto an islet capture plate, and incubating them in a non-CO₂ incubator. We then detail procedures for adding mitochondrial stressor solutions and conducting the mitochondrial stress test using the Seahorse XFe24 Analyzer.

Before you begin

Recent research has revealed the significant role of mitochondria in adipose tissue homeostasis.^2,3 As a gold standard for characterizing mitochondrial function, the mitochondrial stress test using the Seahorse Instrument has emerged as a necessary method for assessing adipose tissue physiological functions and pathological changes. In the past few years, our laboratory has optimized a protocol for performing Seahorse experiments in isolated fat pads from mice and achieved accurate and replicable measurements of adipose mitochondrial respiration. The protocol below describes the specific steps for using ex vivo mouse brown and white adipose depots.

Institutional permissions

Animal experiments should follow ethical guidelines and principles approved by the Institutional Animal Care and Use Committee. All procedures on animals in this study have been approved by the Animal Welfare Committee (AWC), the Institutional Animal Care and Use Committee (IACUC) of The University of Texas Health Science Center at Houston (AWC-21-0075).

The mice were housed in a controlled environment with free access to food and water, adhering to a 12-h light-dark cycle at the University of Texas Health Science Center at Houston’s animal facility, where humidity and temperature (21°C) were regulated. 14-week-old Wild-type (C57BL/6J) male mice were used for this protocol. This protocol should use at least three biological replicates and five technical replicates per condition.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Mouse white adipose depot	This study	N/A
Mouse brown adipose depot	This study	N/A
Chemicals, peptides, and recombinant proteins
XF calibrant medium	Agilent Technologies	100840-000
XF base medium	Agilent Technologies	102353-100
Sodium pyruvate	Gibco	11360-070
L-glutamine	Gibco	A29168-01
Glucose	Fisher Chemical	D16-500
Oligomycin	MilliporeSigma	O4876
FCCP	MilliporeSigma	C2920
Rotenone	MilliporeSigma	557368
Antimycin-A	MilliporeSigma	A8674
DMSO	MilliporeSigma	D8418
NaOH	Fisher Chemical	SS254-4
PBS	Corning	21-040-CV
Experimental models: Organisms/strains
C57BL/6J (male, homozygous, 14 weeks old)	The Jackson Laboratory	Strain#:000664; RRID:IMSR_JAX:000664
Software and algorithms
Wave	Agilent Technologies	https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-software/seahorse-wave-desktop-software-740897
GraphPad Prism 10	GraphPad Software, Inc.	https://www.graphpad.com
Other
Seahorse XFe24 Islet Capture FluxPak	Agilent Technologies	103518-100
Seahorse XFe24 Analyzer	Agilent Technologies	N/A
Scales	OHAUS	PX85
Water bath	Thermo Fisher Scientific	FSGPD10
pH detector	Thermo Fisher Scientific	STARA2140
Spring scissors	VWR	100493-888
Scissors	VWR	82027-578
Forceps	VWR	82027-388
25 mm syringe filter	Fisher Scientific	09719C
150 mL filter system	Corning	431153
6-well plate	Corning	CLS3506
6 cm dish	Corning	CLS430166
Sterile glass 250 mL conical flask	Fisher Scientific	FB501250
25 mL reagent reservoir	Biolog	3102

Materials and equipment

Seahorse assay medium

Seahorse assay medium (for one 24-well plate)
Reagent	Final concentration	Amount
XF base medium	N/A	53.515 mL
Sodium Pyruvate (100 mM)	1 mM	550 μL
L-Glutamine (200 mM)	2 mM	550 μL
Glucose (1 M)	7 mM	385 μL
Total		55 mL

CRITICAL: Commercially available ready-to-use solutions include XF base medium, 100 mM Sodium Pyruvate, and 200 mM L-Glutamine stored at 4°C according to the manufacturer’s instructions. Glucose solutions are prepared using glucose compounds and stored at 4°C after passing through a 25 mm syringe filter (0.2 μm). The Seahorse assay medium needs to be freshly prepared on the day of experiments and kept warm in a 37°C water bath.

Mitochondrial Stress Test Compound solutions

Compound solutions for mitochondrial stress test (for one 24-well plate)
Port	Reagent	Concentration in port	Final concentration	Amount of stock	Total assay medium
A	Oligomycin (10 mM)	20 μM	2.1 μM	6 μL	3 mL
B	FCCP (10 mM)	80 μM	8.4 μM	24 μL	3 mL
C	Rotenone (10 mM)	20 μM	2.6 μM	6 μL	3 mL
	Antimycin-A (40 mM)	40 μM	5.2 μM	3 μL

CRITICAL: All reagents are prepared in the fume hood utilizing appropriate PPE, solubilized to stock concentration with DMSO, aliquoted, and stored at −20°C away from light (up to 12 months). The solutions A, B, and C need to be prepared freshly before the assays under the fume hood and kept warm in a 37°C water bath.

Alternatives: The Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies Catalog #103015-100) is an alternative for compound solution preparation.

Step-by-step method details

The day before the experiment

Timing: 16–24 h

For proper function, the sensors in Seahorse plates must be thoroughly hydrated before use. Steps 1 and 2 are instructions for how to hydrate the sensor cartridge plate.

• 1.
  Rehydrate the sensor plate.

  • a.
    Open the Seahorse XFe FluxPak to retrieve the XFe24 sensor cartridge plate.
  • b.
    Add 1 mL of XF calibrant medium into each well of the bottom utility plate (the upper plate with sensor probes needs to be placed upside down on the lab bench to protect the sensors from damage).
  • c.
    Re-assemble the entire cartridge unit so that all sensors are submerged in the sterile calibrant medium. Please see Figure 1 for a detailed view.

Note: Avoid damaging the sensors on the probe plate during rehydration.

Note: Bubbles may form between the sensor surface and the medium. To remove the bubbles, move the plate up and down slightly.

Note: Verify that the XF Calibrant level is high enough to keep the sensors submerged.

Note: Ensure the pink hydro booster is placed back on top of the utility plate before the rehydration.

• 2.Place the entire cartridge in a CO₂-free cell culture incubator at 37°C for 16–24 h.

Figure 1.

The composition of the XFe24 sensor cartridge plate

The XFe24 sensor cartridge plate consists of a transparent cover lid, a green sensor cartridge, a pink hydro booster, and a transparent utility plate. The sensor cartridge has 24 wells, each with 4 compound ports, named A, B, C, and D.

The day of the experiment

Timing: 20 min (for step 3)

Timing: 5 min (for step 4)

Timing: 90 min (for step 5)

Timing: 20 min (for step 6)

Timing: 180 min (for step 7)

Timing: approximately 30 min (for step 8)

• 3.Prepare the Seahorse assay medium.
  We recommend preparing fresh Seahorse assay medium on the day of the experiment.

  • a.
    Follow the “seahorse assay medium” table in “materials and equipment” to prepare the medium in a sterile glass 250 mL conical flask.
  • b.
    Heat the medium to 37°C in a water bath for 20 min.
  • c.
    Adjust the pH to 7.4 using 1N NaOH.
  • d.
    Sterile-filter the medium using a 150 mL filter system.
  • e.
    Place the medium back in a water bath to keep warm until usage.
• 4.Open one Islet Capture Microplate from the Seahorse XFe FluxPak and hydrate all tissue screens using the XF calibrant medium.
• 5.Prepare the brown adipose tissue (BAT) and subcutaneous white adipose tissue (sWAT) samples.
  To perform the Seahorse Assay on ex vivo adipose tissues, the white and brown adipose depots need to be freshly harvested from mice.

  • a.
    Mice are euthanized by a minimum dosage of inhaled isoflurane followed by a quick cervical dislocation.
  • b.
    The interscapular BAT and sWAT (also identified as inguinal WAT) are carefully removed and placed into one well of 6-well plate with PBS on ice.

    CRITICAL: Avoid contamination from adjacent muscle tissues when collecting sWAT using surgical tools.

    CRITICAL: Avoid contamination from adjacent muscle tissues and surrounding white fat tissues when collecting BAT using surgical tools.

    Note: We further direct readers to two previously published protocol articles for a better demonstration and visualization of harvesting interscapular BAT.^4,5

  • c.
    For each BAT fat pad, cut out five replicate pieces of approximately 3–5 mg; for each sWAT fat pad, cut out five replicate pieces of approximately 5–8 mg.
  • d.
    Weigh each small piece of fat pad using a 6-cm dish in a highly accurate balance (0.01 mg readability). Record the weight of each piece.

    Note: To ensure cutting out of similar-sized tissues, disposable tissue punches (e.g., Biopunch, ID 2.0 mm, 15111-20 or Biopunch, ID 2.5 mm, 15111-25) are recommended.

    Note: Prior to weighing the sample, the samples are slightly placed on tissue to absorb the liquid.

  • e.
    Transfer these small pieces of tissue to the wells of the Islet Capture Microplate and further mince into smaller tissue chunks using a fine scissor. Please see Figure 2 for a detailed view.

    Note: Do not place any tissue samples in the background correction wells: A1, B4, C3, and D6.

    Note: In case more wells are needed to hold tissue samples, a minimum of two background correction wells are required.

  • f.
    Insert tissue screens into each well to keep tissues attached to the bottom (Figure 3).

    Note: Make sure the flat side of each screen faces tissues and tissues are in the center of each well.

    Note: Make sure there is a capture screen in each well, even if there are no tissue samples in the well (such as background correction wells).

    Note: (Optional) A Seahorse XF Islet Capture Screen Insert Tool (101135-100, Agilent Technologies) can be utilized to facilitate the insertion of the tissue screen.

  • g.
    Add 625 μL seahorse medium to each well and incubate in the CO₂-free cell culture incubator at 37°C for 45–60 min.
• 6.Prepare compound solutions for the mitochondrial stress test.
  We recommend preparing reagent stocks before the assay and preparing fresh compound solutions on the day of the experiment.

  • a.
    Follow the “mitochondrial stress test compound solutions” table in “materials and equipment” to dilute the stock compounds oligomycin, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone/antimycin-A into a working concentration using freshly prepared Seahorse assay medium.
  • b.
    Take out the hydrated cartridge plate.
  • c.
    Add freshly made compound solutions A, B, and C into corresponding ports A, B, and C on the cartridge, respectively. For a 625-μL medium volume in the well, we inject 75 μL of solution A (oligomycin), 83 μL of solution B (FCCP), and 93 μL of solution C (rotenone/antimycin-A) to ports A, B, and C, respectively. Please see Figure 4 for an illustration of transfer compounds into ports. For troubleshooting, see problem 1.

    CRITICAL: Refrain from tapping the plate to eliminate air bubbles.

    Note: To prevent the formation of air bubbles, ensure the liquid is dispensed gently to the first stop of the pipette.

    Note: Make sure to adjust the pipetting volume before adding each compound solution into ports.

• 7.Use the WAVE software to control the Seahorse XFe24 Analyzer and run the mitochondrial stress test assays (also called the BOFA (Basal-Oligomycin-FCCP-Antimycin-A) assay). For troubleshooting, see problems 2, 3, 4, and 5.

CRITICAL: We recommend the following setup for adipose tissues:

Figure 2.

Preparation of sWAT and BAT samples

After weighing each tissue piece, samples were transferred to the center of the well, and each small piece was minced 20-30 times until samples became homogenized.

(A) sWAT sample before being minced.

(B) sWAT sample after being minced.

(C) BAT sample before being minced.

(D) BAT sample after being minced. Abbreviation: sWAT: subcutaneous white adipose tissue; BAT: brown adipose tissue.

Figure 3.

Insertion of tissue screens into wells of Islet Capture Microplate

(A) Tissue screens in the package.

(B) Hydrate tissue screens in Seahorse XF Calibrant to remove any bubbles.

(C) Hold the edge of the tissue screen when the flat side faces down.

(D) Insert the tissue screen all the way into the bottom of wells containing tissues in the center (samples) or without tissues (background correction wells).

(E) Ensure each screen is firmly clicked in without any movement in each well.

Figure 4.

Addition of compound solutions into the ports on the sensor cartridge

Prepare compound solutions freshly, transfer each solution to a sterile reagent reservoir, and add each compound solution to its corresponding port (Port A, B, or C) using a multi-channel pipette. Port D will be utilized when an additional compound is needed.

Oligomycin: 3 cycles, mix 3 min, wait 2 min, measure 4 min;

FCCP: 3 cycles, mix 3 min, wait 2 min, measure 2 min;

Rotenone/antimycin-A: 3 cycles, mix 3 min, wait 2 min, measure 4 min.

Note: This protocol is not intended to show a tutorial for using the WAVE software. Please read details into how to navigate the WAVE software through this user guide: https://www.agilent.com/cs/library/usermanuals/public/S7894-10000_Rev_C_Wave_2_6_User_Guide.pdf and the protocol by Mayberry et al.⁶

• 8.
  Analysis of the results.

  • a.
    When the assay is completed, the results can be exported into Excel or GraphPad for further analysis.
  • b.
    Normalize the results to tissue weights. Please see Figure 5 for an example.

    Note: Protein concentration is an alternative normalization method. Extract the protein from the tissues in each well and normalize the result to the protein concentration.

    Note: More examples can be found in our recently published articles.^1,7

  • c.
    The statistical method of Two-way ANOVA is recommended.

Figure 5.

The mitochondrial respiration results of ex vivo sWAT and BAT

Here, we collected the sWAT and BAT from 14-week-old C57BL/6J male mice (n = 2) and followed this protocol to run the Seahorse mitochondrial stress test assay. The result was normalized to the tissue weight, showing that BAT displayed higher mitochondrial respiration than WAT. Abbreviation: sWAT: subcutaneous white adipose tissue; BAT: brown adipose tissue. For each mouse, five technical replicates were included. Data: mean ± SEM. Two-Way ANOVA.

Expected outcomes

A reduction of oxygen consumption rate (OCR) is expected in response to oligomycin (Complex V inhibitor) injection, an increase of OCR will be observed following FCCP (uncoupler) injection, and an inhibition of mitochondrial respiration is expected upon injection of rotenone (Complex I inhibitor) and antimycin-A (Complex III inhibitor). For the example shown in Figure 5, the BAT is expected to display higher OCR values than WAT with the same weights.

Limitations

Consideration of sex dimorphism, aging, and diet-induced obesity. It has been found that the expression of mitochondrial genes in adipose tissue varies in different sexes.⁸ Therefore, this protocol was developed based on samples from male mice and might need to be further optimized when experiments are conducted in female mice. Similarly, considering that aged mice and diet-induced obese mice displayed decreased mitochondrial content in adipose tissues,^9,10,11 it is beneficial to adjust the protocol by increasing the size of adipose tissue depots and/or optimizing mitochondrial stressor concentrations to accommodate age- or diet-induced mitochondrial alterations.

Expanded applications to other tissues. This protocol is considered suitable for adipose depots, and when applied to other tissues, different substrate concentrations, reagent dosages, tissue sizes, and measurement times need to adapt to tissue types and their metabolic activities.

Uncovered cell-type-specific mitochondrial activity. Considering that adipose tissue contains not only adipocytes but also other cells, such as immune cells, endothelial cells, etc., this protocol using ex vivo fat pads reflects an overview of mitochondrial respiration at the tissue level and is not aiming to differentiate cell-type-specific mitochondrial activity.

Troubleshooting

Problem 1

Bubbles in cartridge plate ports after adding compound solutions (Step 6c).

Potential solutions

• •Use a fine needle to remove any visible bubbles in the port.

Problem 2

No response to compounds (Step 7).

Potential solutions

• •Use freshly prepared compounds to treat the tissues (Steps 3 and 6).
• •Tissue preparation time should be kept short to maintain normal mitochondrial viability (Step 5).
• •Perform pilot experiments to titrate and optimize compound concentrations.
• •During the assay, a longer period of Wait and Measure is needed for oligomycin, and for FCCP, a shorter period of Measure is recommended (Step 7).

Problem 3

Too low or too high OCR value at baseline level (Step 7).

Potential solutions

• •The weight of the fat tissue samples should be controlled within the recommended weight range (Step 5c).
• •For BAT, substrate (glucose) elevation in the Seahorse assay medium may increase baseline OCR values (Step 3).

Problem 4

High variations among depots in technical replicates for each biological replicate (Step 7).

Potential solutions

• •The weights of the fat tissue samples in each group should be similar (Step 5c).
• •Each tissue piece should be taken from a fixed location of fat pads, e.g., near the lymph nodes of sWAT (Step 5c).
• •Use disposable tissue punches to ensure comparable size of fat tissue samples (Step 5c).

Problem 5

Inconsistency among biological replicates (Step 7).

Potential solutions

• •Use sex- and age-matched mice and littermate controls. If special diets are utilized, diet content and duration need to be consistent.
• •Keep consistency in harvesting locations, tissue sizes, and tissue weights (Step 5c).
• •Increase biological replicate number to minimize the impact by variations within the same biological group.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Yu A. An (Yu.An@uth.tmc.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Dr. Fenfen Wang (Fenfen.Wang@uth.tmc.edu).

Materials availability

This study did not generate new unique reagents or materials.

Data and code availability

This study did not generate any unique datasets or code.