Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Summary

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Abstract

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Introduction

Exosomes are the smallest subset of EVs, 50–200 nm in diameter, and have multiple biological functions across a diverse array of signaling processes^1,2,3,4,5. They govern cellular and tissue homeostasis primarily by facilitating intercellular communication through cargo molecules such as lipids, proteins, and nucleic acids^6,7,8,9. One critical step in EV research is the isol....

Protocol

The utilization of animals and all animal procedures were approved by the Institutional Animal Care and Use Committees (IACUC) at Cedars-Sinai Medical Center (CSMC).

1. Murine Bronchoalveolar Lavage Fluid (BALF) Collection and Preparation

1. BALF collection
   1. Euthanize mice with a cocktail of ketamine (300 mg/kg) and xylazine (30 mg/kg) via the intraperitoneal route followed by cervical dislocation.
   2. Insert a 22 G angiocatheter into the trachea. Attach an insulin syringe containing 1 mL (mL) of ice-cold sterile Dulbecco’s phosphate buffer saline (DPBS) and instill 1 mL of DBPS into both lungs through the a....

Results

We performed EV isolation from mouse BALF using UFC and UC-DGC isolation methods on the same day. The UFC method required approximately 2.5–3 h, whereas the UC-DGC technique required 8 h of processing time. This did not include buffers and reagent preparation time. It should be noted that some other tasks could be performed during the long centrifugation periods. Nevertheless, the entire procedure lasted nearly an entire day for the UC-DGC isolation technique.

Discussion

In the past few decades, scientists have unraveled the significances of EVs in cellular homeostasis. More importantly, EVs play major roles in many disease processes by modulating neighboring and distant cells through their bioactive cargo molecules^{1,21,22,26,27,28,29,}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
Amicon Ultra-15 centrifugal filters Ultracel-100K	Sigma-Millipore, St. Louis, MO	UFC910024
Dulbecco's Phosphate Buffered Saline (DPBS)	Corning Cellgro, Manassas, VA	21-031-CV
Sucrose	Sigma-Millipore, St. Louis, MO	EMD8550
HEPES	Research Products International, Prospect, IL	75277-39-3
EDTA	Corning Cellgro, Manassas, VA	46-034-CI
Sodium Chloride	Sigma-Millipore, St. Louis, MO	S3014-1KG
OptiPrep	Sigma-Millipore, St. Louis, MO	MKCD9753	Density Gradient Medium
Ketamine	VetOne, Boise, ID	13985-702-10
Xylazine	Akorn Animal Health, Lake Forest, IL	59399-110-20
Syringe 1 mL	BD Syringe, Franklin Lakes, NJ	309656
Angiocatheter 20 G	BD Syringe, Franklin Lakes, NJ	381703
Centrifuge tubes 15 mL	VWR, Radnor, PA	89039-666
Centrifuge tubes 50 mL	Corning Cellgro, Manassas, VA	430828
Bicinchonic acid (BCA) protein assay	Pierce, Thermo Fischer Scientific, Rockford, IL	23235
Rabbit anti-mouse TSG101 Antibody	AbCam, Cambridge, MA	AB125011
Rat anti-mouse PE-CD63 Antibody	Biolegend, San Diego, CA	143904
CD81
CD9
Anti-rabbit IgG, HRP-linked antibody	Cell Signaling Technology, Danvers, MA	7074S
4x LDS
10x Reducing agent (Bolt)
10x Lysis buffer (Bolt)	Cell Signaling Technology, Danvers, MA
Bolt 4-12% Bis-Tris Plus acrylamide gel	Invitrogen, Thermo Fisher Scientific, Waltham, MA	NW04120
iBlot 2 Nitrocellulose mini stacks	Invitrogen, Thermo Fisher Scientific, Waltham, MA	IB23002
Chemiluminescent HRP antibody detection reagent HyGLO	Denville Scientific, Holliston, MA	E2400
Ultracentrifuge tubes 17 mL	Beckman Coulter, Pasadena, CA	337986
Ultracentrifuge tubes 38.5 mL	Beckman Coulter, Pasadena, CA	326823
Corning SFCA Syringe Filters 0.2 µm pore	Thermo Fisher Scientific, Waltham, MA	09-754-13
Equipment
Centrifuge	Eppendorf, Hamburg, Germany	-
Ultracentrifuge	Beckman Coulter, Pasadena, CA	-
Nanosight (NS300)	Malvern, Worcestershire, UK	-	To measure particle size distribution and particle concentration
MACSQuant Analyzer 10 flow cytometer	Miltenyi Biotec, Bergisch Gladbach, Germany	-
iBlot Transfer Apparatus	Thermo Fischer Scientific, Waltham, MA	-
Bio-Rad ChemiDoc MP Imaging System	Bio-Rad, Hercules, CA
FlowJo v. 10			Analysis software