# Protocol for isolating and characterizing effector functions of murine bone marrow-derived eosinophils following bacterial challenge

## TL;DR

This paper provides a detailed protocol for isolating and studying mouse bone marrow-derived eosinophils in response to bacterial infection.

## Contribution

A novel protocol for isolating and characterizing murine eosinophils' effector functions after bacterial challenge.

## Key findings

- Steps for differentiating bone marrow progenitor cells into eosinophils are described.
- Functional assays for cytotoxicity, bactericidal activity, and cytokine production are outlined.
- The protocol uses a Bordetella bronchiseptica model adaptable to other microorganisms.

## Abstract

Understanding eosinophil-associated mechanisms, especially in the context of infection, has been steadily increasing. Here, we present a protocol for isolating and differentiating murine bone marrow-derived eosinophils (bmEos) followed by multi-well plate assays to evaluate eosinophilic responses to bacterial challenge. We describe steps for evaluating eosinophil effector functions including cytotoxicity, bactericidal activity, and cytokine production through multiplex ELISA panels. We use a Bordetella bronchiseptica infection model, but this approach can be modified for an array of microorganisms and different experimental settings.

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

•Steps for differentiating mouse bone marrow progenitor cells into eosinophils (bmEos)•Instructions on analyzing purity of differentiated bmEos via flow cytometry•Guidance on preparing B. bronchiseptica inoculums for bmEos infection assays•Procedures for functional in vitro assays following bmEos bacterial challenge

Steps for differentiating mouse bone marrow progenitor cells into eosinophils (bmEos)

Instructions on analyzing purity of differentiated bmEos via flow cytometry

Guidance on preparing B. bronchiseptica inoculums for bmEos infection assays

Procedures for functional in vitro assays following bmEos bacterial challenge

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

Understanding eosinophil-associated mechanisms, especially in the context of infection, has been steadily increasing. Here, we present a protocol for isolating and differentiating murine bone marrow-derived eosinophils (bmEos) followed by multi-well plate assays to evaluate eosinophilic responses to bacterial challenge. We describe steps for evaluating eosinophil effector functions including cytotoxicity, bactericidal activity, and cytokine production through multiplex ELISA panels. We use a Bordetella bronchiseptica infection model, but this approach can be modified for an array of microorganisms and different experimental settings.