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Updated: Jun 4, 2025
A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
Published on: March 10, 2017
A protocol for high-throughput screening for small chemicals promoting macrophage-mediated tumor cell phagocytosis in
Zehua He1, Ziyi Hu2, Lei Wang2
1Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing, China; Shanghai Frontiers Science Center for Drug Target Identification and Delivery, and the Engineering Research Center of Cell and Therapeutic Antibody of the Ministry of Education, National Key Laboratory of Innovative Immunotherapy, School of Pharmaceutical Sciences, Shanghai Jiao Tong University, Shanghai, China.
Abstract:
Macrophage-mediated phagocytosis has emerged as a pivotal mechanism for eliminating tumor cells within the realm of cancer immunotherapy. Here, we present a protocol for identifying small molecules that enhance phagocytosis in mice using a co-culture system comprising primary macrophages, cancer cells, and a blockade of phagocytic checkpoints. We describe steps for expressing enhanced green fluorescent protein-luciferase (eGFP-Luc) and producing bone marrow-derived macrophages (BMDMs). We then detail procedures for optimizing co-culture conditions for high-throughput screen (HTS) and executing HTS chemical identification. For complete details on the use and execution of this protocol, please refer to Cao et al. 1.
Insights
This study details a protocol to discover small molecules that boost macrophage phagocytosis for cancer immunotherapy. The method uses a co-culture system to identify compounds enhancing tumor cell elimination by macrophages.
Area of Science:
- Immunology
- Cancer Biology
- Drug Discovery
Background:
- Macrophage phagocytosis is crucial for cancer immunotherapy.
- Identifying agents that enhance phagocytosis is a key goal.
Purpose of the Study:
- To present a protocol for identifying small molecules that enhance macrophage phagocytosis of tumor cells.
- To enable high-throughput screening (HTS) for novel immunomodulatory compounds.
Main Methods:
- Development of a co-culture system with primary macrophages and cancer cells.
- Expression of enhanced green fluorescent protein-luciferase (eGFP-Luc) in cancer cells.
- Optimization of co-culture conditions for HTS and chemical identification.
- Inclusion of phagocytic checkpoint blockade in the screening system.
Main Results:
- The protocol enables systematic identification of small molecules enhancing phagocytosis.
- The described methods facilitate the discovery of potential cancer immunotherapy agents.
Conclusions:
- This protocol provides a robust platform for discovering phagocytosis-enhancing small molecules.
- The findings support the development of novel immunotherapies targeting macrophage-mediated tumor cell clearance.
