# Protocol for isolating leukemia-derived extracellular vesicles from the spleen of preclinical models of leukemia using ultracentrifugation

## TL;DR

This paper provides a detailed protocol for isolating small extracellular vesicles from the spleen of leukemia mouse models to study their role in disease progression and biomarker discovery.

## Contribution

A novel protocol for isolating leukemia and microenvironment-derived extracellular vesicles from murine spleens using ultracentrifugation.

## Key findings

- The protocol efficiently enriches leukemia-derived extracellular vesicles and those from the tumor microenvironment.
- Characterization methods include immunoblotting, microscopy, size determination, and flow cytometry.
- The approach supports investigations into extracellular vesicle roles in leukemia progression and biomarker identification.

## Abstract

Here, we present a protocol for the direct isolation of small extracellular vesicles (sEVs) from the spleen of preclinical murine models of leukemia using ultracentrifugation. We describe steps for tissue collection, sample preparation, ultracentrifugation-based isolation, and sEV characterization. This protocol allows for efficient enrichment of both leukemia and its microenvironment-derived sEV (LME-sEV), providing a valuable tool for studying their composition and functional roles. Potential applications include investigating the role of sEV in leukemia progression and identifying biomarkers.

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

•Isolation of sEV from the spleen of preclinical murine models of leukemia•Full representation of sEV produced in the tumor microenvironment•Characterization by immunoblotting, microscopy, size determination, and flow cytometry

Isolation of sEV from the spleen of preclinical murine models of leukemia

Full representation of sEV produced in the tumor microenvironment

Characterization by immunoblotting, microscopy, size determination, and flow cytometry

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

Here, we present a protocol for the direct isolation of small extracellular vesicles (sEVs) from the spleen of preclinical murine models of leukemia using ultracentrifugation. We describe steps for tissue collection, sample preparation, ultracentrifugation-based isolation, and sEV characterization. This protocol allows for efficient enrichment of both leukemia and its microenvironment-derived sEV (LME-sEV), providing a valuable tool for studying their composition and functional roles. Potential applications include investigating the role of sEV in leukemia progression and identifying biomarkers.