# Protocol for in vivo lineage tracing of regeneration-associated macrophages from injured skeletal muscle of adult mice

## TL;DR

This paper describes a method to track macrophage changes during skeletal muscle regeneration in mice using genetic labeling and cell transplantation.

## Contribution

A novel in vivo lineage tracing protocol for regeneration-associated macrophages using genetic labeling and FACS-isolated cell transplantation.

## Key findings

- Macrophages from CD45.1+ donors were transplanted into injured CD45.2+ mouse muscles and tracked over time.
- The protocol includes steps for muscle injury, cell isolation, transplantation, and phenotyping during regeneration.
- Flow cytometry was used to phenotype transplanted macrophages at specific regenerative time points.

## Abstract

Macrophages undergo phenotypic transitions that are essential for successful skeletal muscle (SkM) regeneration. Here, we present a protocol for in vivo lineage tracing of regeneration-associated macrophages, combining genetic labeling with transplantation of fluorescence-activated cell sorting (FACS)-isolated cells. Macrophages isolated from congenic CD45.1+ donor mice are transplanted into pre-injured SkMs of CD45.2+ mice and phenotyped by flow cytometry at designated time points of the regenerative process. We describe steps for muscle injury, SkM tissue processing, macrophage isolation, transplantation, and flow cytometry phenotyping.

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

•Injury and conditioning of skeletal muscle of donor and recipient mice for transplant•Instructions for isolating regeneration-associated macrophages by FACS•Steps for transplanting individual macrophage subpopulations into recipient muscles•Procedures for phenotyping transplanted macrophages in recipient muscles

Injury and conditioning of skeletal muscle of donor and recipient mice for transplant

Instructions for isolating regeneration-associated macrophages by FACS

Steps for transplanting individual macrophage subpopulations into recipient muscles

Procedures for phenotyping transplanted macrophages in recipient muscles

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

Macrophages undergo phenotypic transitions that are essential for successful skeletal muscle (SkM) regeneration. Here, we present a protocol for in vivo lineage tracing of regeneration-associated macrophages, combining genetic labeling with transplantation of fluorescence-activated cell sorting (FACS)-isolated cells. Macrophages isolated from congenic CD45.1+ donor mice are transplanted into pre-injured SkMs of CD45.2+ mice and phenotyped by flow cytometry at designated time points of the regenerative process. We describe steps for muscle injury, SkM tissue processing, macrophage isolation, transplantation, and flow cytometry phenotyping.