Related Experiment Video
Updated: Jul 11, 2025
Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System
Published on: October 20, 2023
Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages
Uener Ribeiro Dos Santos1, Julyanna Oliveira de Castro1, Melissa Ercília Santos Matos1
1Laboratory of Immunobiology - Department of Biological Sciences, State University of Santa Cruz - UESC.
Abstract:
Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the fungal clearance efficiency of macrophages.
Insights
This study details a method to analyze how macrophages engulf and clear fungal conidia. These techniques help measure fungal clearance efficiency, crucial for understanding immune responses to fungal pathogens.
Area of Science:
- Immunology
- Mycology
- Cell Biology
Background:
- Macrophages are key immune cells defending against pathogens.
- Fungal conidia phagocytosis is vital for studying host-pathogen interactions and fungal virulence.
- Understanding fungal clearance mechanisms is essential for controlling infections.
Purpose of the Study:
- To present a comprehensive protocol for analyzing the phagocytosis, clearance, and viability of *T. stromaticum* conidia by macrophages.
- To provide methods for evaluating macrophage-mediated fungal clearance efficiency.
- To facilitate research on immune responses against pathogenic fungi.
Main Methods:
- *Trichoderma* culture and conidia preparation.
- Isolation of human peripheral blood mononuclear cells (PBMCs) and differentiation into macrophages.
- In vitro phagocytosis assay using stained conidia and coverslips.
- Clearance assay to determine conidia viability post-phagocytosis.
Main Results:
- The described protocol enables the quantitative analysis of conidial phagocytosis by macrophages.
- The methods allow for the assessment of fungal conidia viability after being engulfed by macrophages.
- The study establishes techniques to measure the efficiency of macrophage-mediated fungal clearance.
Conclusions:
- The presented methods offer a robust framework for investigating macrophage-fungal interactions.
- This protocol is valuable for studying fungal virulence factors and host immune evasion strategies.
- The techniques can be applied to assess the efficacy of antifungal immune responses.
