TY  - JOUR
TI  - Extraction and Isolation of Extracellular Vesicles From Piper betle Leaves Using the Apoplastic Fluid Washing and Size Exclusion Chromatography Method
AU  - Sanddy, Izy 
AU  - Adawiyah, Wan Rahimatul
AU  - Ooi, Der Jiun
AU  - Ekanayake, Gayandi 
AU  - Fazeli, Alireza 
AU  - Rahman, Noor Izzah Abd
AU  - Khan, Norhayati Liaqat Ali
VL  - 16
IS  - 9
PY  - 2026
DA  - 2026/05/05
SP  - e5685
C1  - Bio-protocol 2026;16:e5685
DO  - 10.21769/BioProtoc.5685
UR  - https://doi.org/10.21769/BioProtoc.5685
AB  - Plant-derived extracellular vesicles (PDEVs) have emerged as important mediators of intercellular communication and hold growing potential in therapeutic applications. However, standardized methods for their isolation, particularly from Piper betle leaves (PBL), remain unexplored. Existing apoplastic fluid washing (AFW) extraction techniques typically rely on manual syringe infiltration, which often leads to inconsistent pressure control, variable yields, and increased risk of tissue damage. This protocol describes a vacuum-assisted AFW extraction method optimized for the recovery of intact extracellular vesicles (EVs) from PBL. The workflow features controlled negative pressure using a vacuum pump and chamber to achieve more efficient leaf infiltration compared to infiltration using the syringe method and reproducible apoplastic fluid (AF) collection with subsequent low-speed centrifugation steps, to ensure minimal contamination and preservation of vesicle integrity. Piper betle–derived extracellular vesicle (PBdEV) isolation and purification steps are performed using size exclusion chromatography (SEC). The size and concentration of PBdEVs were confirmed using nanoparticle tracking analysis (NTA), whereas the cup-shaped and lipid bilayer morphology of the EVs were confirmed using transmission electron microscopy (TEM). The method is scalable and adaptable to various leaf morphologies and physiological states, making it suitable for both exploratory and high-throughput studies. Overall, this protocol provides a more consistent, efficient, and tissue-preserving alternative to traditional syringe-based AF extraction methods, offering higher-quality EV preparations for plant EV research.
KW  - Apoplastic fluid washing
KW  - Piper betle leaves
KW  - Extracellular vesicles
KW  - Piper betle–derived extracellular vesicles
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False