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Published on: September 22, 2013
A Multi-Fluorophore Staining Scheme for Identification and Quantification of Vomocytosis
Noah Pacifici1, Tatu Rojalin1, Randy P Carney1
1Department of Biomedical Engineering, University of California-Davis, Davis, California 95616, United States.
Abstract:
Vomocytosis is a process by which fungal pathogens, for instance, Cryptococcus neoformans (CN), escape from the digestive phagolysosome of phagocytic cells after ingestion. Interestingly, this expulsion leaves both the pathogen and phagocyte unharmed, and is believed to be an important mechanism by which CNs disseminate throughout infected hosts. This phenomenon was discovered in 2006, and research to date has relied almost entirely on quantification via manual counting of vomocytosis events in time-lapse microscopy videos. This archaic method has the significant disadvantages of requiring excessive labor in manual analysis, limited throughput capabilities, and low accuracy due to subjectivity. Here, we present an alternative method to measure vomocytosis rates using a multi-fluorophore reporter system comprised of two in situ staining steps during infection and a flow cytometry readout. This approach overcomes the limitations of conventional time lapse microscopy methods, with key advantages of high throughput capability, simple procedural steps, and accurate objective readouts. This study rigorously characterizes this vomocytosis reporter system in CN-infected MΦ and DC cultures via fluorescence microscopy, confocal microscopy, and flow cytometry. Here, this fluorescent tool is used to observe differences in expulsion rates after phagosome-modifying drug treatments and additionally utilized to distinguish differences in biochemical compositions among fluorescence-activated cell sorted fungal populations via Raman spectroscopy. Furthermore, this reporter scheme is demonstrated to be adaptable for use in measuring potential biomaterial particle expulsion events. Ultimately, the fluorescent reporter system presented here provides a universal tool for vomocytosis rate measurement of phagocytosed material. This facile approach opens the door to previously unfeasible types of vomocytosis-related studies such as high throughput treatment mechanistic screening and downstream characterization of expelled material.
Insights
Researchers developed a new fluorescent reporter system to accurately measure vomocytosis, a fungal pathogen expulsion process. This high-throughput method replaces laborious manual counting, enabling new avenues for studying fungal dissemination and drug screening.
Area of Science:
- Mycology
- Immunology
- Cell Biology
Background:
- Vomocytosis is a mechanism used by fungal pathogens like Cryptococcus neoformans (CN) to escape phagocytic cells.
- Current methods for quantifying vomocytosis rely on manual counting from time-lapse microscopy, which is labor-intensive, low-throughput, and subjective.
- Accurate measurement of vomocytosis is crucial for understanding fungal dissemination and developing effective treatments.
Purpose of the Study:
- To develop and characterize a novel, high-throughput method for measuring vomocytosis rates.
- To overcome the limitations of traditional manual counting techniques.
- To provide a universal tool for quantifying the expulsion of phagocytosed materials.
Main Methods:
- A multi-fluorophore reporter system involving two in situ staining steps during infection.
- Flow cytometry for objective and high-throughput quantification of vomocytosis events.
- Validation using fluorescence microscopy, confocal microscopy, and Raman spectroscopy in macrophage (MΦ) and dendritic cell (DC) cultures.
Main Results:
- The novel reporter system provides accurate, objective, and high-throughput measurement of vomocytosis rates.
- The system successfully quantified differences in expulsion rates following phagosome-modifying drug treatments.
- Demonstrated adaptability for measuring biomaterial particle expulsion and analyzing expelled fungal populations.
Conclusions:
- The developed fluorescent reporter system offers a facile and universal tool for measuring vomocytosis.
- This method significantly improves upon conventional techniques, enabling previously infeasible research.
- Facilitates high-throughput screening of treatments and downstream analysis of expelled materials.
