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A new and simple method for studying the binding and ingestion steps in the phagocytosis of yeasts
J Giaimis1, Y Lombard, M Makaya-Kumba
1Université Louis Pasteur, Départment d'Immunologie et Immunopharmacologie, Illkirch, France.
Abstract:
Autoclaved yeasts are stained light pink by May-Grünwald Giemsa (MGG). If treated with tannic acid solution just before MGG staining, they display a deep violet color. It seemed possible that these properties could be used to discriminate between extra- and intracellular yeasts in a phagocytosis test, extracellular yeasts being violet and intracellular yeasts being pink. To validate this protocol, quantitative studies of phagocytosis by MALU cells (a murine macrophage cell line) were performed in the presence or absence of drugs known to interfere with phagocytosis. After treatment of cells with cytochalasin B, the mean number of pink yeasts per cell decreased in a dose-dependent manner, the mean number of violet yeasts increased in a dose-dependent manner, whereas the total number of cell-associated yeasts remained almost unchanged whatever the dose used. After treatment with alpha-mannans or chloroquine, the mean numbers of both violet and pink yeasts decreased in a dose-dependent manner. These results confirmed that (i) violet yeasts are extracellular, (ii) autoclaved yeasts recognize lectin receptors, and (iii) unstained (pink) yeasts are intracellular. We show that this simple method can be used for quantitative light microscopic analysis of both the attachment and internalization steps in the phagocytosis of yeasts.
Insights
A new staining method distinguishes extracellular (violet) from intracellular (pink) yeasts during phagocytosis. This technique allows for quantitative analysis of yeast-macrophage interactions, aiding in the study of cellular uptake mechanisms.
Area of Science:
- Immunology
- Cell Biology
- Microscopy
Background:
- Phagocytosis is a critical cellular process for immune defense.
- Distinguishing between extracellular and intracellular pathogens is essential for understanding immune responses.
- Current methods for analyzing phagocytosis can be complex and time-consuming.
Purpose of the Study:
- To develop and validate a simple, quantitative staining method for differentiating extracellular and intracellular yeasts during phagocytosis.
- To confirm the reliability of the staining method using pharmacological inhibitors of phagocytosis.
Main Methods:
- Autoclaved yeasts were stained with May-Grünwald Giemsa (MGG) alone (pink) or pre-treated with tannic acid before MGG staining (violet).
- Murine macrophage-like cells (MALU) were incubated with yeasts in the presence or absence of phagocytosis inhibitors (cytochalasin B, alpha-mannans, chloroquine).
- Quantitative light microscopy was used to count pink (intracellular) and violet (extracellular) yeasts associated with cells.
Main Results:
- Violet yeasts consistently represented extracellular yeasts, while pink yeasts represented intracellular yeasts.
- Inhibitors of phagocytosis (cytochalasin B, alpha-mannans, chloroquine) affected the number of pink and violet yeasts in a dose-dependent manner, validating the method.
- The total number of cell-associated yeasts remained stable when phagocytosis was inhibited, indicating accurate discrimination.
Conclusions:
- The tannic acid-enhanced MGG staining protocol provides a reliable and simple method for distinguishing extracellular and intracellular yeasts.
- This technique enables quantitative light microscopic analysis of yeast attachment and internalization during phagocytosis.
- The method confirms that autoclaved yeasts interact with lectin receptors on macrophages.