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Cellular Autofluorescence in Mouse Embryonic Fibroblasts Interferes with Antigen Detection Using Flow Cytometry
Selcen Çelik-Uzuner1,2, Chris O'Neill3
1Human Reproduction and Development Unit, Kolling Institute for Medical Research, Sydney Medical School, University of Sydney, Sydney, 2065, Australia. selcen.celik@ktu.edu.tr.
Journal of Fluorescence
|March 27, 2021
Summary
Cellular autofluorescence in mouse embryonic fibroblasts can interfere with immunostaining analysis. This study highlights the need for rigorous controls to ensure accurate quantitative results in flow cytometry and fluorescence microscopy.
Area of Science:
- Cell Biology
- Immunology
- Biotechnology
Background:
- Immunostaining is a key method for analyzing cellular markers.
- Flow cytometry offers quantitative analysis of large cell populations.
- Standard protocols involve fixation, permeabilization, blocking, and antibody staining.
Purpose of the Study:
- To investigate cellular autofluorescence in mouse embryonic fibroblasts.
- To assess the impact of autofluorescence on immunostaining.
- To emphasize the importance of controls in quantitative analysis.
Main Methods:
- Immunostaining of mouse embryonic fibroblasts.
- Analysis using flow cytometry.
- Evaluation of autofluorescence across multiple fluorescence channels.
Main Results:
- High levels of cellular autofluorescence were detected in mouse embryonic fibroblasts.
- Autofluorescence interfered with the detection of cellular antigens using common fluorophores.
- Useful analysis was restricted to a single fluorescence channel due to autofluorescence.
Conclusions:
- Cellular autofluorescence presents a significant limitation for immunostaining techniques.
- Thorough controls are essential for specific and quantitative analysis.
- Researchers must be aware of potential autofluorescence interference in their experiments.
