# Isolation of Intestinal Macrophage Subpopulations for High-Quality Total RNA Purification in Zebrafish

## TL;DR

This paper introduces a method to isolate and study different types of intestinal macrophages in zebrafish using a fluorescent marker and RNA analysis.

## Contribution

A novel FACS-based strategy for isolating intestinal macrophage subpopulations and extracting high-quality RNA in zebrafish.

## Key findings

- A FACS strategy was developed to isolate intestinal macrophage subpopulations in zebrafish based on GFP and morphology.
- High-quality RNA was successfully purified from each macrophage subpopulation for transcriptomic analysis.
- The method enables molecular characterization of macrophage subpopulations to study their functional roles.

## Abstract

Intestinal macrophages have been poorly studied in fish, mainly due to the lack of specific molecular markers for their identification and isolation. To address this gap, using the zebrafish Tg(mpeg1:EGFP) transgenic line, we developed a fluorescence-activated cell sorting strategy (FACS) that allows us to isolate different intestinal macrophage subpopulations, based on GFP expression and morphological differences. Also, we achieved the purification of high-quality total RNA from each population to perform transcriptomic analysis. The complete strategy comprises three steps, including intestine dissection and tissue dissociation, the isolation of each intestinal macrophage population via FACS, and the extraction of total RNA. To be able to characterize molecularly different macrophage subpopulations and link them to their functional properties will allow us to unravel intestinal macrophage biology.