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Published on: October 18, 2016
An Imaging Flow Cytometry Protocol for Studying Immunoregulatory Receptor-Mediated Regulation of Phagocytosis
Myron A Zwozdesky1, Chenjie Fei1, James L Stafford2
1Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada.
Insights
This study details a method using imaging flow cytometry to analyze how immune cell receptors interact. It measures receptor crosstalk
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry is crucial for studying innate immune cell functions.
- Imaging flow cytometry integrates microscopy with high-throughput data acquisition.
- Understanding receptor crosstalk is key to regulating immune responses.
Purpose of the Study:
- To describe a method for co-expressing stimulatory and inhibitory immunoregulatory receptors.
- To measure receptor crosstalk in the regulation of phagocytosis.
- To provide guidance on reagent selection and instrument calibration.
Main Methods:
- Co-expression of specific immunoregulatory receptor types in AD293 cells.
- Utilizing imaging flow cytometry for high-resolution cellular analysis.
- Applying automated template analyses for data interpretation.
Main Results:
- Successful co-expression of diverse receptor types was achieved.
- Receptor crosstalk significantly influenced phagocytic activity.
- The method allows for detailed quantitative analysis of cellular responses.
Conclusions:
- The described method enables robust investigation of immunoregulatory receptor interactions.
- Imaging flow cytometry is a powerful tool for dissecting complex cellular signaling pathways.
- This approach can advance the study of innate immunity and phagocytosis regulation.
Abstract:
Advances in flow cytometry have allowed for innovative functional investigations of innate immune cell responses. Imaging flow cytometers combine the imaging capabilities of microscopy with rapid, high-throughput data acquisition attributes of standard flow cytometers. Here, we describe a detailed method for co-expressing stimulatory and inhibitory immunoregulatory receptor-types in AD293 cells and then measuring receptor cross-talk during the regulation of the phagocytic response. Information on reagent selection, imaging flow cytometry calibration, and automated template analyses are included.
