Using flow cytometry to measure phagocytic uptake in earthworms

Area of Science:

• Immunology
• Cell Biology
• Zoology

Background:

• Phagocytosis is a critical innate immune mechanism in invertebrates like earthworms.
• Earthworm coelomocytes (leukocytes) are key immune cells involved in engulfing pathogens.
• Flow cytometry offers a powerful tool for quantitative analysis of cellular functions.

Purpose of the Study:

• To familiarize students with flow cytometry principles and applications.
• To develop quantitative reasoning skills through data analysis of cellular assays.
• To investigate phagocytosis by earthworm coelomocytes using fluorescent bacteria.

Main Methods:

• Isolation of coelomocytes (hyaline amoebocytes, granular amoebocytes, chloragocytes) from Eisenia hortensis.
• In vitro incubation of coelomocytes with fluorescent Escherichia coli to assess phagocytosis.
• Flow cytometry analysis using fluorescence and scatter properties to quantify phagocytic activity.
• Gating strategies to analyze specific coelomocyte subsets.

Main Results:

• Successful isolation and characterization of earthworm coelomocytes.
• Quantification of phagocytosis levels by different coelomocyte populations.
• Demonstration of flow cytometry's utility in dissecting immune cell function.
• Students developed proficiency in data acquisition and analysis.

Conclusions:

• The laboratory module effectively integrates flow cytometry techniques with innate immunity research.
• Students acquire practical skills in cellular assays, data analysis, and scientific reporting.
• This module provides a foundational understanding of immune mechanisms and quantitative biology.