Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Melissa J. Grimm; Anthony C. D'Auria; Brahm H. Segal

doi:10.3791/51678

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

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09:04 min

July 9, 2014

DOI: 10.3791/51678-v

Melissa J. Grimm¹, Anthony C. D'Auria¹, Brahm H. Segal²

¹Department of Medicine,Roswell Park Cancer Institute, ²Department of Medicine, School of Medicine and Biomedical Sciences,University of Buffalo

Overview

This study presents a method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores using confocal microscopy. The procedure involves labeling macrophages, harvesting them, and challenging them with fungal spores to assess their phagocytic capabilities.

Key Study Components

Area of Science

Immunology
Microbiology
Cell Biology

Background

Alveolar macrophages play a crucial role in the immune response.
Understanding their interaction with pathogens like Aspergillus is vital for developing therapeutic strategies.
Confocal microscopy allows for detailed visualization of cellular processes.
This study aims to elucidate the mechanisms of macrophage-mediated pathogen control.

Purpose of Study

To evaluate the effectiveness of alveolar macrophages in controlling fungal growth.
To utilize confocal microscopy for real-time observation of macrophage interactions with pathogens.
To contribute to the understanding of immune responses to fungal infections.

Methods Used

Labeling of alveolar macrophages with a lipophilic dye (PKH 26).
Harvesting macrophages via bronchoalveolar lavage from inoculated mice.
Culturing and collecting GFP positive fungal spores.
Challenging macrophages with GFP positive candida and observing at predetermined time points.

Main Results

Confocal microscopy revealed the dynamics of macrophage-fungal interactions.
Isolated macrophages demonstrated varying abilities to control fungal growth.
The study provided insights into the phagocytic efficiency of alveolar macrophages.
Results may inform future research on immune responses to fungal infections.

Conclusions

Alveolar macrophages can effectively control the growth of phagocytosed Aspergillus spores.
Confocal microscopy is a valuable tool for studying macrophage functions.
This method can be applied to further investigate immune responses in various contexts.

Frequently Asked Questions

What is the significance of using confocal microscopy?

Confocal microscopy allows for high-resolution imaging of cellular interactions in real-time, providing insights into the dynamics of immune responses.

How are alveolar macrophages harvested?

Macrophages are harvested from inoculated mice using bronchoalveolar lavage, a technique that collects cells from the lungs.

What role do alveolar macrophages play in the immune system?

Alveolar macrophages are essential for the clearance of pathogens and play a key role in the lung's immune defense.

Why is it important to study fungal infections?

Fungal infections can be severe and are often difficult to treat; understanding immune responses can lead to better therapeutic strategies.

What are GFP positive fungal spores?

GFP positive fungal spores are genetically modified to express green fluorescent protein, allowing for easy tracking during experiments.

What are the potential applications of this research?

This research can inform therapeutic approaches for fungal infections and enhance understanding of macrophage biology.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The overall goal of this procedure is to compare the ability of macrophages to control the growth of ceto fungal candia ex vivo. This is accomplished by first labeling the alveolar macrophages in vivo with a lipophilic dye PKH 26. The second step of the procedure is to harvest alveolar macrophages from the inoculated mice by bronchoalveolar lavage.

The third step is to culture and collect GFP positive fungal spores. The final steps are to challenge macrophages with GFP positive candia and fixed cells at predetermined time points. Ultimately, with minimal ex vivo cell manipulation, confocal microscopy can reveal the ability of alveolar macrophages to control the growth of phagocytose.

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