# Protocol for measuring endogenous 24(S),25-epoxycholesterol and inducing innate immune memory in mouse macrophages

## TL;DR

This paper provides a detailed protocol for measuring a specific cholesterol metabolite and inducing immune memory in mouse macrophages.

## Contribution

A new protocol for quantifying 24(S),25-epoxycholesterol and inducing trained immunity in mouse macrophages is introduced.

## Key findings

- A workflow for precise quantification of 24(S),25-epoxycholesterol using LC-MS is described.
- Procedures for establishing metabolite-induced trained immunity in BMDMs are detailed.
- ATAC-seq and cytokine assays are outlined for evaluating immune memory.

## Abstract

Multiple metabolic pathways and metabolites are involved in innate immune memory induction of macrophages; however, protocols for in vitro-trained immunity assays induced by metabolites in mouse macrophages are limited. Here, we present a protocol for measuring endogenous 24(S),25-epoxycholesterol and inducing innate immune memory in mouse macrophages. We describe steps for sample preparation, measurement of 24(S),25-epoxycholesterol, and establishment of an in vitro-trained immunity model. We then detail procedures for assays measuring cytokine concentration and for assay for transposase-accessible chromatin using sequencing (ATAC-seq).

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

•Instructions on culturing and differentiation of bone marrow-derived macrophages (BMDMs)•Workflow for precise quantification of endogenous 24(S),25-EC using LC-MS•Validation procedures for establishing metabolite-induced trained immunity in BMDMs

Instructions on culturing and differentiation of bone marrow-derived macrophages (BMDMs)

Workflow for precise quantification of endogenous 24(S),25-EC using LC-MS

Validation procedures for establishing metabolite-induced trained immunity in BMDMs

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

Multiple metabolic pathways and metabolites are involved in innate immune memory induction of macrophages; however, protocols for in vitro-trained immunity assays induced by metabolites in mouse macrophages are limited. Here, we present a protocol for measuring endogenous 24(S),25-epoxycholesterol and inducing innate immune memory in mouse macrophages. We describe steps for sample preparation, measurement of 24(S),25-epoxycholesterol, and establishment of an in vitro-trained immunity model. We then detail procedures for assays measuring cytokine concentration and for assay for transposase-accessible chromatin using sequencing (ATAC-seq).