Protocol for isolating and characterizing extracellular vesicles by ultracentrifugation from bone marrow-derived macrophages

Summary

Extracellular vesicles (EVs) enable the transmission of crucial molecular components between the parental and recipient cells. Macrophages can polarize into two distinct macrophage phenotypes, thereby exerting diverse effects on recipient cells. Here, we present a protocol for the direct isolation of EVs from bone marrow-derived macrophages (BMDMs) using ultracentrifugation. We describe steps for culturing BMDMs and macrophage polarization. We then detail procedures for further characterizing the isolated EVs using transmission electron microscopy (TEM), western blotting, and nanoparticle tracking analysis (NTA).

Graphical abstract

Highlights

• •Steps for extracting bone marrow-derived macrophages (BMDMs)
• •Instructions for EV isolation from BMDMs by ultracentrifugation
• •Procedures for EV characterization by TEM, western blotting, and NTA

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

Extracellular vesicles (EVs) enable the transmission of crucial molecular components between the parental and recipient cells. Macrophages can polarize into two distinct macrophage phenotypes, thereby exerting diverse effects on recipient cells. Here, we present a protocol for the direct isolation of EVs from bone marrow-derived macrophages (BMDMs) using ultracentrifugation. We describe steps for culturing BMDMs and macrophage polarization. We then detail procedures for further characterizing the isolated EVs using transmission electron microscopy (TEM), western blotting, and nanoparticle tracking analysis (NTA).

Before you begin

Extracellular vesicles (EVs) from bone marrow-derived macrophages (BMDMs) are considered crucial carriers that mediate the immunoregulatory functions of macrophages, influence the microenvironment, and participate in intercellular communication. Investigating the effects of EVs from BMDMs under different polarization states on downstream cells (such as other immune cells, tumor cells, endothelial cells, etc.) is crucial for elucidating the pathophysiological mechanisms related to macrophages. Here, we provide a comprehensive protocol for isolating EVs by ultracentrifugation from mouse bone marrow-derived macrophages, followed by further characterization of the isolated EVs using transmission electron microscopy, western blotting, and nanoparticle tracking analysis. We have applied this protocol to directly isolate EVs from BMDMs using ultracentrifugation and characterize them. This protocol can also be utilized for isolating and characterizing EVs derived from other cell types. Furthermore, the generation and culture of BMDMs in this protocol are equally suitable for investigating the roles of macrophages in various diseases.

Institutional permissions

Please ensure that animal experiments are conducted in accordance with the ethical standards for the care of animals. The protocol used in this study has been authorized by the Animal Care and Use Committee of Nanjing Medical University. All animals were housed in the Specific Pathogen-Free (SPF) Animal Center of Nanjing Medical University.

Preparation of extracellular vesicle-depleted fetal bovine serum

Timing: 120 min

• 1.Heat-inactivate fetal bovine serum (FBS) by incubating in a 56°C water bath for 30 min.
• 2.Prepare extracellular vesicles (EV)-depleted FBS by centrifugation at 100,000 × g for 120 min at 4°C using 70PC bottle ultracentrifuge tubes with a P45AT fixed-angle rotor.

Note: Steps 1 and 2 must not be interchanged. Otherwise, protein aggregates potentially formed during heat inactivation may interfere with subsequent EV isolation.

• 3.Collect the supernatant and sterilize using 0.22 μm syringe filter.

Note: Extracellular vesicles (EV)-depleted FBS can be used for the preparation of DMEM medium.

Preparation of reagents, tools, and equipment

Timing: 120 min

• 4.Prepare sterile surgical scissors, forceps, and microcentrifuge tubes through autoclaving.
• 5.Filter DMEM medium and sterile phosphate-buffered saline (PBS) through a 0.22 μm filter and store at 4°C before use.
• 6.Before the start of the experiment, preheat DMEM medium and sterile PBS in a 37°C water bath.

Note: To eliminate the potential effect of temperature on macrophage polarization.

• 7.Pre-cooling centrifuges and their rotors.
• 8.
  Dissolve macrophage colony-stimulating factor (M-CSF).

  • a.
    Before opening, centrifuge the vial containing M-CSF lyophilized powder at 1000 × g for 5 min at 4°C.

    Note: Allow the lyophilized powder adhering to the wall or cap to gather at the bottom of the vial to facilitate dissolution.

  • b.
    Reconstitute to a concentration of 0.1–0.5 mg/mL in sterile distilled water or sterile PBS.

    Note: Avoid vortex or vigorously pipetting the protein.

  • c.
    Pipette gently several times and then let it stand for 5 min.
  • d.
    Dispense the recombinant protein solution into autoclaved microcentrifuge tubes to reduce freeze/thaw cycles. Store in a −20°C refrigerator for up to 3 months.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-CD63 antibody (1:1,000 dilution)	Abcam	Cat # ab217345; RRID: AB_2754982
Anti-GM130 antibody (1:500 dilution)	BD Biosciences	Cat # 610823; RRID: AB_398142
Anti-TSG101 antibody (1:2,000 dilution)	Abcam	Cat # ab133586; RRID: AB_2943043
Anti-rabbit IgG-HRP conjugate antibody (1:5,000 dilution)	Cell Signaling Technology	Cat # 7074S; RRID: AB_2099233
Anti-mouse IgG-HRP conjugate antibody (1:5,000 dilution)	Cell Signaling Technology	Cat # 7076S; RRID: AB_330924
Chemicals, peptides, and recombinant proteins
Recombinant mouse CSF-1/M-CSF protein	ABclonal	Catalog # RP01216
Certified fetal bovine serum	VivaCell	Catalog # C04001-500
DMEM high-glucose medium	VivaCell	Catalog # C3113-0500
PBS, 1× (phosphate-buffered saline)	Servicebio	Catalog # G4202-500ML
Penicillin-streptomycin solution	VivaCell	Catalog #C3420-0100
Lipopolysaccharide (LPS) solution	eBioscience	Catalog # 00-4976-93
Recombinant mouse/rat mature TGF-beta 1 protein	ABclonal	Catalog # RP00671
SDS-PAGE sample loading buffer, 5×	Biosharp	Catalog # BL502B
20× TBST buffer	Solarbio	Catalog # T1082
Skim milk powder	Biosharp	Catalog # BS102-500g
Critical commercial assays
Pierce BCA protein assay kit	Thermo Fisher Scientific	Catalog # 23225
Experimental models: Organisms/strains
Mouse: C57BL/6 wild-type, 8–10 weeks old, males and females	Specific pathogen-free laboratory animal center of Nanjing Medical University	N/A
Other
Cell culture dish 100 × 20 mm	Bioland	Catalog # CCD06-100A
50 mL centrifuge tube	Corning	Catalog # 430829
Red blood cell lysis buffer	Beyotime	Catalog # C3702-120ml
0.22 μm sterile syringe filter	Millipore	Catalog # SLGPR33RB
70 μm cell strainers	Bioland	Catalog # CS05-070
10 mL serological pipets	Corning	Catalog # 4488
1 mL disposable sterile syringe	Kindly Group	N/A
Allegra X-15R centrifuge	Beckman Coulter	N/A
Centrifuge	Baiyang	Catalog # BY-R18
Ultracentrifuge	Hitachi	Catalog # CP100NX
70PC bottle ultracentrifuge tube	Himac	Catalog # S308433A
P45AT fixed-angle rotor	Himac	Catalog # 5720211109
Transmission electron microscope	Hitachi	Catalog # HT-7700
Carbon-coated copper grids	Beijing Aou Qihang Technology Co., Ltd.	Catalog # H11032
PVDF membrane	Millipore	Catalog # IPFL00010
Chemiluminescent imaging device	Tanon	Catalog # 5200

Materials and equipment

Here, we describe the media components used to culture bone marrow-derived macrophages.

BMDMs media

Reagent	Stock concentration	Final concentration	Amount
DMEM high glucose medium	N/A	N/A	445 mL
Certified Fetal Bovine Serum	N/A	10%	50 mL
Penicillin-Streptomycin Solution	10,000 U/mL	100 U/mL	5 mL
Total		N/A	500 mL

Store at 4°C for up to 14 days.

Premix all of the above BMDMs media additives and sterilize them through a 0.22 μm filtration system. When preparing the culture medium, it is important to use EV-depleted FBS. FBS contains EVs, which can contaminate cell-derived EVs and may interfere with downstream analysis.¹

Step-by-step method details

Generation of BMDMs

Timing: 7 days

This step details how to generate bone marrow-derived macrophages (BMDMs) (Figure 1).

• 1.Euthanize C57BL/6 mice (approximately 8–10 weeks old), spray 75% ethanol on the abdomen and lower regions of the mice, and place in a supine position. Troubleshooting.
• 2.Eliminate the muscles and connective tissue carefully following separating the whole leg bones with scissors.

Note: Gauze can be used to assist in the separation of the connective tissue and muscles of the leg bones.

• 3.Use scissors to gently cut along the area above the femoral greater trochanter and below the ankle joint.
• 4.Cut the knee joint, separate the tibia and femur.

Note: Do not break the bones in steps 3 and 4, maintain the integrity of the bone marrow cavity.

• 5.Add an appropriate amount of 75% ethanol to a 10 cm diameter cell culture dish in advance, transfer the bones into it, and soak for 5 min.
• 6.Take another 10 cm cell culture dish and add 10 mL DMEM medium (without FBS) into it.
• 7.After rinsing the tibia and femur with an appropriate amount of DMEM medium (without FBS) (Figure 2A), place the bones into the dish from step 6.
• 8.Cut both ends of the bones and use a 1 mL syringe to aspirate an appropriate amount of DMEM medium without FBS to infuse the marrow cavity (Figure 2B).

Note: Each bone needs to be infused multiple times until it gradually turns white.

• 9.Pass the medium containing bone marrow cells from the 10 cm cell culture dish through a 70 μm cell strainer and centrifuge at 1500 × g for 5 min.
• 10.Discard the supernatant, resuspend the cells in 4 mL red blood cell lysis buffer, mix by pipetting, and incubate for 4 min.
• 11.After incubation, add 4 mL of DMEM medium without FBS and centrifuge at 1500 × g for 5 min to terminate the lysis process of the red blood cell lysis solution.
• 12.Discard the supernatant and resuspend the cells using DMEM medium with 10% FBS.
• 13.Transfer the cell suspension from Step 12 to a new 10 cm cell culture dish and adjust the total volume to 10 mL using DMEM medium with 10% FBS.
• 14.Place in a cell culture incubator for 2 h.
• 15.After 2 h later, gently aspirate the medium from the 10 cm cell culture dish into a 50 mL sterile centrifuge tube.

CRITICAL: Avoid repeated blow-sucking in the 10 cm cell culture dish to prevent interference with the settling effect, as monocytes are characterized by low density and delayed adherence.²

• 16.According to the cell density, replenish the 50 mL centrifuge tube with an appropriate amount of DMEM medium with 10% FBS to adjust the cell suspension to 1–2 × 10⁵ cells/mL. Then add M-CSF to the tube and mix by pipetting.

Note: The final concentration of M-CSF in the centrifuge tube should be 30 ng/mL.

• 17.Remove 10 mL of cell suspension from the centrifuge tube in step 16 to a 10 cm cell culture dish and place it into a cell culture incubator.
• 18.Three days later, discard the medium in the dish. Add 10 mL DMEM medium (10% FBS and 30 ng/mL M-CSF) to the cells.
• 19.After 6–7 days of differentiation, collect cells for further use.

Note: After 6–7 days of differentiation, one side of the tibia and femur from a mouse can yield approximately 0.5–1.5 × 10⁷ BMDMs.

CRITICAL: Flow cytometry analysis can be recommended to demonstrate the effectiveness of the differentiation into BMDMs.

Figure 1.

Generation of BMDMs overview

Illustrative BMDMs generation overview (by Figdraw).

Figure 2.

Treatment of leg bones

(A) Rinse the tibia and femur with an appropriate amount of DMEM medium (without FBS).

(B) Cut both ends of the bones and use a 1 mL syringe to infuse the marrow cavity.

Polarization of macrophages

Timing: 1 day

Macrophages exhibit remarkable plasticity. Various stimulation conditions, such as oxidized phospholipids, hypoxic environments, and metabolic products, can induce macrophages to polarize into distinct macrophage phenotypes with unique markers and functional characteristics, thereby exerting diverse effects on recipient cells. Among these, classically activated macrophages (M1) and alternatively activated macrophages (M2) represent the most classical and extensively studied subtypes. This section focuses on the M1/M2 classification to describe the polarization of macrophages.

• 20.
  If the cell confluency of BMDMs cultured for 6–7 days in step 19 reaches approximately 70%–80%, rinse with sterile 37°C PBS after discarding the medium in the dish.

  • a.
    Preheat sterile PBS in a 37°C water bath.
  • b.
    Aspirate and discard the medium (containing 10% FBS and 30 ng/mL M-CSF) from the 10 cm cell culture dish.
  • c.
    Add PBS and repeat the washing step 2–3 times.

Note: Perform rinsing gently to avoid cell detachment.

• 21.Add 10 mL of preheated (37°C) DMEM medium (without FBS) to each 10 cm cell culture dish and incubate in a cell culture incubator for a minimum of 6 h to as long as 12 h.

Note: The length of serum starvation can vary depending on the cell sensitivity to nutrient deprivation.

• 22.After sufficient starvation, discard the medium from the dish. Add 10 mL DMEM medium (without FBS) supplemented with either M1 polarization agents (30 ng/mL M-CSF and 100 ng/mL LPS) or M2 polarization agents (30 ng/mL M-CSF and 2 ng/mL TGFβ1).
• 23.After 24 h of treatment, the cells are collected for further use.

EV isolation

Timing: 4–6 h

In this part of the protocol, differential centrifugation and ultracentrifugation are used to isolate EVs from BMDMs. The principle involving separating EVs, cellular components, and impurity particles is to exploit key parameters, including size and density.

• 24.Allow the centrifuge to cool to 4°C.
• 25.Discard the medium from the polarized macrophages, replace it with fresh DMEM medium with 10% EV-depleted FBS, and culture for 24 h. The medium is collected in a 50 mL centrifuge tube and centrifuged at 300 × g at 4°C for 10 min to precipitate the cells. Troubleshooting.
• 26.Gently transfer the supernatant to a new 50 mL centrifuge tube and discard the pellet.

Note: It is recommended to use a pipette to transfer the supernatant and avoid touching the pellet.

• 27.Centrifuge the supernatant at 2000 × g for 15 min at 4°C.

CRITICAL: This step aims to maximally remove dead cells and cell debris.

• 28.Carefully transfer the supernatant to a new centrifuge tube and discard the pellet.
• 29.Centrifuge the supernatant at 12,000 × g for 30 min at 4°C.

CRITICAL: Be careful when transferring the supernatant in steps 26 and 28. It is advisable to retain a small volume of supernatant above the pellet.

• 30.Filter the collected supernatant through a 0.22 μm filter and transfer the filtrate to a 70PC bottle ultracentrifuge tube.

Note: Rinse the ultracentrifuge tubes with water before use. Subsequently, wash three times with ddH₂O. Soak the ultracentrifuge tubes in 75% ethanol for 10 min, then allow them to dry.

• 31.Centrifuge the supernatant in a pre-cooled P45AT fixed-angle rotor of ultracentrifuge at 100,000 × g for 120 min.

Note: Ultracentrifuge tubes must be precisely balanced. Unbalanced rotors can cause inability to operate or cause damage to the rotors/ultracentrifuge.

• 32.Discard the supernatant and resuspend the pellet in 10 mL PBS. Troubleshooting.

CRITICAL: It is recommended to gently aspirate the supernatant using a pipette to avoid disturbing the pellet.

• 33.Centrifuge the sample at 100,000 × g for 120 min at 4°C.
• 34.Carefully aspirate the supernatant and resuspend the pellet in 100 μL sterile pre-cooled PBS.

Note: The specific buffer and volume for EV resuspension can be adjusted as appropriate.

• 35.Prepare 20 μL of the resuspension for transmission electron microscopy and immunoblotting, and 10 μL for nanoparticle tracking analysis.
• 36.Store the remaining EV resuspension at −80°C for up to 3 months, avoiding repeated freeze/thaw cycles to prevent sample degradation.

Note: Within 3 months, the integrity, average diameter, concentration and other characteristics of extracellular vesicles remain comparable to freshly isolated samples.

EV characterization

Timing: 1 day

In this section, EV samples can be stained and imaged by transmission electron microscopy (TEM). The specific biomarkers of EVs can be verified by western blot.

Note: Do not vortex; use a pipette to handle the EVs solution instead.

Pause point: EVs can be characterized according to the following methods.

• 37.Fix the EVs with 1 mL of 2% paraformaldehyde for 5 min.
• 38.Process the copper grid with glow discharge for 1 min.
• 39.Add 10 μL of EVs suspension to the copper grid and incubate for 1 min.

Note: The sample can be diluted to 1–10 × 10⁹ according to the actual concentration of EVs in advance.

• 40.Use filter paper to remove the excess EVs suspension.
• 41.Pipet 10 μL of uranyl acetate solution onto the grid surface and incubate for 1 min.
• 42.Use filter paper to remove the excess uranyl acetate solution.
• 43.Dry for 3 min at 25°C.
• 44.Observe by TEM at 100 kV.
• 45.Obtain the TEM imaging results. Troubleshooting.
• 46.
  Western blotting analysis:

  • a.
    Quantify the protein concentration of EVs using the BCA Protein Assay Kit and normalize the concentration. Mix each sample with 5× loading buffer and incubate them at 95°C for 5 min.
  • b.
    Perform standard SDS-PAGE electrophoresis to separate proteins based on their molecular weight.
  • c.
    Transfer proteins onto the PVDF membrane at 100 V for 80 min.
  • d.
    Block the membrane with 5% milk in TBST for 1 h.
  • e.
    Incubate the membrane with CD63 antibody (1:1,000 dilution) or GM130 antibody (1:500 dilution) or TSG101 antibody (1:2,000 dilution) diluted in TBST for 12 h at 4°C.
  • f.
    Wash the membrane for 15 min with TBST and repeat this step twice.
  • g.
    Incubate the membrane with secondary antibody (1:5,000 dilution) diluted in TBST for 1 h at 25°C.
  • h.
    Wash the membrane for 15 min with TBST and repeat this step twice.
  • i.
    Incubate the blot with the enhanced chemiluminescence (ECL) reagent for 1 min.
  • j.
    Use a chemiluminescent imaging device (Tanon-5200) detect the membrane.

CRITICAL: Nanoparticle tracking analysis can also be recommended for EV characterization to demonstrate the particle size and concentration of EVs.

Expected outcomes

Here, a detailed experimental protocol for the isolation and characterization of extracellular vesicles from mouse bone marrow-derived macrophages (BMDMs). BMDMs are extracted from the leg bones of mice and cultured. Figure 3 confirms the effectiveness of the differentiation into BMDMs. The cell culture medium of BMDMs is collected for centrifugation and ultracentrifugation, and then EVs are isolated from it. We recommend the use of further characterization analysis to evaluate the presence, purity, particle size, and concentration of the EV samples to further determine the validity and reproducibility of this protocol. The morphology of purified EVs from BMDMs can be characterized by transmission electron microscopy (TEM) (Figure 4). Figure 5 provides a western blotting image, confirming the presence of EVs in the sample. The particle size and concentration of EVs derived from BMDMs are characterized by Nanoparticle Tracking Analysis (NTA). The measured size distribution of EVs is shown in Figure 6. It is worth noting that although we have used this protocol for studies mainly in BMDMs, our protocol is expected to be applicable to other types of cells as well.

Figure 3.

Flow cytometry analysis of BMDMs

Representative flow plot identifying the differentiation of BMDMs analyzed by flow cytometry.

Figure 4.

TEM image of EVs from BMDMs

EVs separated from BMDMs were imaged by TEM. Scale bar: 200 nm.

Figure 5.

Western blotting analysis of EV biomarkers

The biomarkers of EVs from BMDMs were analyzed by western blot (TSG101, GM130, and CD63).

Figure 6.

Nanoparticle tracking analysis (NTA) of the EVs

The size and concentration of EVs derived from BMDMs were characterized by NTA.

The International Society for Extracellular Vesicles (ISEV) updated the relevant guidelines for EVs in 2023, and it can provide new theoretical support for EVs research.³

Limitations

Ultracentrifugation for extracellular vesicle (EV) isolation is an effective and reproducible strategy. However, compared to alternative isolation techniques such as size-exclusion chromatography and ultrafiltration,⁴ the yield of EVs is relatively low, and constrained by rotor specifications and tube capacities in ultracentrifugation systems. The quantity of bone marrow-derived macrophages (BMDMs) correlates with the number of leg bones, thereby restricting the application of ultracentrifugation. The culture time of BMDMs increases the time needed for EV extraction. Additionally, the duration required for EV isolation by ultracentrifugation further prolongs the overall process. In addition, each EV isolation method inherently contains a certain amount of contaminants, and ultracentrifugation is no exception.^5,6

Troubleshooting

Problem 1

Low yield of BMDMs (steps 1–19).

Potential solution

The quantity of BMDMs correlates with the number of leg bones. If the yield of BMDMs is low, the number of leg bones used for BMDM generation should be appropriately increased. In the process of taking mouse leg bones, the integrity of the bone marrow cavity should be maintained to avoid some leg bones being unavailable. In Step 8, when perfusing the bone marrow cavity with a 1 mL syringe, the cavity should be repeatedly flushed until the bone gradually turns white to fully collect bone marrow-derived cells.

Problem 2

Low yield of EVs (steps 24–36).

Potential solution

Among the factors affecting the yield and enrichment of EVs, the number of cell culture dishes is the most critical. The number of cells used for EV isolation can be increased by increasing the number of cell culture dishes.

Problem 3

After ultracentrifugation of the supernatant, the EVs pellet is mistakenly aspirated and discarded (steps 32–34).

Potential solution

Mark the side of the ultracentrifuge tube to clearly indicate the position of the pellet, and aspirate the supernatant from the opposite side of the tube. The action should be gentle, avoiding rapid and large movements of the ultracentrifuge tube.

Problem 4

The images of TEM are not clear (step 45).

Potential solution

If the background of the TEM results is dark, the staining time with uranyl acetate solution should be shortened, and 1 min of staining is usually sufficient. It is recommended to treat the copper grid by glow discharge before using the TEM to increase the hydrophilicity of the grid surface and the uniformity of the sample. This helps obtain clearer and more accurate images and improves the reliability and repeatability of the detection.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Zeping Gui (gzp@njmu.edu.cn).

Technical contact

Operational inquiries regarding protocol implementation should be answered by the technical contact, Yuxi Chen (yuxichen2000@163.com).

Materials availability

This study did not produce novel reagents.

Data and code availability

This study did not generate datasets/code.

Acknowledgments

This research was supported by the Natural Science Foundation of China (82470790 and 82170769 to M.G. and 81900684 to Z.W.), the Special Fund for Science and Technology Program of Jiangsu Province (Key Research and Development Plan for Social Development Project) (BE2023784 to M.G.), Jiangsu Province Natural Science Foundation Program (BK20191063 to Z.W.), and Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX25_0803 to R.D.).

Author contributions

Z.G., M.G., S.S., and Z.W. conceived the project. Y.C., R.D., J.M., B.W., and J.X. conducted the experiments. Y.C. wrote the manuscript.

Declaration of interests

The authors declare no competing interests.