Related Experiment Video
Updated: Aug 9, 2026
Flow Cytometry Analysis of Immune Cells Within Murine Aortas
Published on: July 1, 2011
Assessment of endothelial immunophenotype--limitation of flow cytometric analysis
1Department of Immunology, Princess Margaret Hospital, Perth, Western Australia.
Insights
Flow cytometry may underestimate endothelial antigen expression when cells are suspended. Microscopic analysis offers better morphological insights, suggesting caution with flow cytometry for endothelial antigen quantification.
Area of Science:
- Cell Biology
- Immunology
- Microscopy Techniques
Background:
- Flow cytometry is standard for quantifying cell surface antigens in suspension.
- Endothelial cells grow as monolayers, requiring dissociation for flow cytometry or alternative methods like immunoperoxidase staining.
Purpose of the Study:
- To evaluate the accuracy of flow cytometry for quantifying endothelial antigens.
- To compare flow cytometry with immune microscopy for assessing endothelial antigen expression and morphology.
Main Methods:
- Endothelial cells were analyzed using flow cytometry after dissociation.
- Immune microscopy was employed to visualize antigen distribution and morphology.
- Comparison of antigen quantification between flow cytometry and image analysis of microscopy.
Main Results:
- Flow cytometry underestimated the expression of certain antigens on endothelial cells.
- Morphological details of antigens like serpins and fibronectin were only visible via immune microscopy.
- Immune microscopy, while subjective, provided crucial morphological data absent in flow cytometry.
Conclusions:
- Caution is advised when using flow cytometry to quantify endothelial antigens due to potential underestimation.
- Immune microscopy offers superior morphological insights into endothelial antigens.
- Computerized image analysis of immunostaining may mitigate the subjectivity of traditional immune microscopy.
Abstract:
Flow cytometry is generally utilized to quantify antigen expression by cells in suspension. To detect antigens on endothelium, which grows as a monolayer, either the creation of a suspension of endothelial cells for flow cytometry, or the use of alternative techniques (such as immunoperoxidase staining) is required. We demonstrate here that creating suspensions of endothelial cells for flow cytometry underestimates the expression of certain antigens. In addition, morphological information regarding certain antigens (serpins and fibronectin) is only discernible by immune microscopy, a subjective procedure. We would recommend caution in using flow cytometry for the estimation of endothelial antigens. Using computerized estimates of microscopic immunostaining (e.g., with video image analysis) it may be possible to overcome some of the subjective limitations of immune microscopy.
