[Sorting of living and dead cells by flow cytometry--staining principle and basic experiment]

Area of Science:

• Cell biology
• Cytometry
• Biochemistry

Background:

• Distinguishing between living and dead cells is crucial in various biological and medical research fields.
• Traditional methods for cell viability assessment can be time-consuming or lack precision.
• Flow cytometry offers a high-throughput method for cellular analysis.

Purpose of the Study:

• To describe a reliable method for simultaneous identification of living and dead cells.
• To validate the use of fluorescein diacetate (FDA) and propidium iodide (PI) staining for cell viability assessment.
• To demonstrate the application of this method in analyzing uterine squamous epithelium samples.

Main Methods:

• Simultaneous staining of cells with fluorescein diacetate (FDA) and propidium iodide (PI).
• Utilizing intracellular esterase activity to generate green fluorescence in living cells (FDA).
• Utilizing DNA intercalation to generate red fluorescence in dead cells (PI).
• Analysis of stained cells using flow cytometry to generate cytograms.

Main Results:

• FDA is hydrolyzed by living cells, producing intracellular green fluorescence.
• PI enters dead cells, intercalates with DNA, and emits bright red fluorescence.
• Flow cytometry clearly distinguishes between green-fluorescent (living) and red-fluorescent (dead) cells.
• In normal uterine squamous epithelium, white blood cells were identified as viable, while superficial and intermediate cells were non-viable.

Conclusions:

• Simultaneous FDA/PI staining provides a robust method for differentiating live and dead cells.
• Flow cytometry analysis of FDA/PI stained cells allows for accurate cell viability assessment.
• This technique is applicable to biological samples such as uterine squamous epithelium, revealing differential cell viability.