Simultaneous three color and electronic cell volume analysis with a single UV excitation source

Area of Science:

• Biomedical Engineering
• Cell Biology
• Analytical Chemistry

Background:

• Flow cytometry is crucial for analyzing cell populations.
• Simultaneous measurement of multiple parameters enhances analytical depth.
• Existing methods may have limitations in analyzing complex cellular heterogeneity.

Purpose of the Study:

• To adapt an Ortho ICP-22 flow cytometer for simultaneous measurement of three fluorochromes and electronic cell volume (ECV).
• To develop a system for analyzing tumor cell and macrophage heterogeneity and differentiation.
• To demonstrate the utility of this system in identifying specific cellular activities and markers.

Main Methods:

• Addition of a third photomultiplier tube to the flow cytometer.
• Development of a new electronic cell volume (ECV) flow cell with a 100 µm aperture.
• Utilizing a 365 nm mercury arc beam for fluorochrome excitation and a delay circuit for signal association.
• Flexible stain combinations for enzyme activities, DNA/RNA, and phagocytic probes.

Main Results:

• Successful simultaneous measurement of three fluorochromes and ECV.
• Demonstrated identification of plasminogen activator-like activity in tumor and inflammatory cells.
• Quantitative expression of macrophage differentiation markers was achieved.
• The system proved valuable for analyzing cellular heterogeneity and differentiation.

Conclusions:

• The adapted flow cytometer provides a flexible and powerful tool for multiparametric cell analysis.
• This technique is effective for studying tumor cell and macrophage heterogeneity and differentiation.
• The system is applicable to various cell sorters and stain combinations, offering broad research utility.