# Protocol for analyzing T cell and macrophage motility in a mouse pancreatic cancer spheroid model using light-sheet microscopy

## TL;DR

This paper provides a detailed protocol for studying how T cells and macrophages move in a mouse pancreatic cancer model using light-sheet microscopy.

## Contribution

The paper introduces a new experimental protocol for analyzing immune cell motility in a pancreatic cancer spheroid model.

## Key findings

- The protocol includes steps for co-culturing macrophages and T cells with pancreatic cancer spheroids.
- Light-sheet microscopy is used to capture time-lapse imaging of immune cell migration.
- Detailed procedures for generating murine bone marrow-derived macrophages and cytotoxic T lymphocytes are provided.

## Abstract

The interaction of macrophages and T cells is pivotal for shaping the immune response to cancer. Here, we present a protocol for investigating the co-culture of macrophages and T cells in a pancreatic cancer spheroid model. We describe steps for analyzing migration patterns by light-sheet microscopy, generating murine bone marrow-derived macrophages (BMDMs) and cytotoxic T lymphocytes (CTLs), and staining CTLs for image analysis. We then detail procedures for generating spheroids using a mouse pancreatic cancer cell line.

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

•Co-culture of BM-derived macrophages and T cells in a pancreatic cancer spheroid model•Detailed instructions for the formation of murine pancreatic cancer spheroids•Time-lapse imaging of T cells and macrophages using light-sheet microscopy

Co-culture of BM-derived macrophages and T cells in a pancreatic cancer spheroid model

Detailed instructions for the formation of murine pancreatic cancer spheroids

Time-lapse imaging of T cells and macrophages using light-sheet microscopy

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

The interaction of macrophages and T cells is pivotal for shaping the immune response to cancer. Here, we present a protocol for investigating the co-culture of macrophages and T cells in a pancreatic cancer spheroid model. We describe steps for analyzing migration patterns by light-sheet microscopy, generating murine bone marrow-derived macrophages (BMDMs) and cytotoxic T lymphocytes (CTLs), and staining CTLs for image analysis. We then detail procedures for generating spheroids using a mouse pancreatic cancer cell line.