Work

Protocol for rapid evaluation of antibody-mediated protection in macrophages using immunofluorescence techniques

Year: 2024

Type: article

Abstract: Antibodies have been shown to provide protection against tuberculosis (TB). It is important to identify the qualitative and quantitative protective effects of antibodies before a clinical study. Here, we present a protocol to evaluate antibody-mediated protection in macrophages using confocal fluorescence microscopy and flow cytometry. We describe steps for bacteria and macrophage preparation, cell infection, phagocytosis, and phagosome maturation analysis. For complete details on the use and execution of this protocol, please refer to Zeng et al. 1 • Steps for infecting RAW264.7 cells with FITC-labeled Bacillus Calmette-Guérin (BCG) • Steps for identifying macrophage phagocytosis efficiency of BCG-FITC by flow cytometry • Details on determining phagosome maturation in BCG-FITC-infected macrophages Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Antibodies have been shown to provide protection against tuberculosis (TB). It is important to identify the qualitative and quantitative protective effects of antibodies before a clinical study. Here, we present a protocol to evaluate antibody-mediated protection in macrophages using confocal fluorescence microscopy and flow cytometry. We describe steps for bacteria and macrophage preparation, cell infection, phagocytosis, and phagosome maturation analysis.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103324

Authors: Lingyuan Zeng, Hao Li

Institution: China Agricultural University

Language: English

Topic: Tuberculosis Research and Epidemiology

Subfield: Infectious Diseases

Field: Medicine

Domain: Health Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,500