Fluorescent dyes for cell viability: an application on prefixed conditions.

Area of Science:

• Cell Biology
• Biotechnology
• Microscopy

Background:

• Apoptosis plays crucial roles in pathological, developmental, and homeostatic processes.
• Fluorescent dyes Acridine Orange (AO), Hoechst 33342 (HO), and Propidium Iodide (PI) are vital for analyzing cell viability.
• These dyes exhibit specificity for live, apoptotic, and late apoptotic/necrotic cells, respectively.

Purpose of the Study:

• To investigate the applicability of HO, AO, and PI for analyzing cell viability in prefixed cell monolayers.
• To describe metachromatic effects observed with dual and triple dye combinations using fluorescence microscopy.
• To propose a simplified staining protocol for identifying cell viability states in fixed cells.

Main Methods:

• Utilized three common cell lines for experiments.
• Applied fluorescence microscopy to observe metachromatic effects of dye combinations.
• Developed and tested a sequential staining method using HO, AO, and PI on prefixed cells.
• Employed a long-pass FITC filter for visualization.

Main Results:

• Demonstrated successful application of HO, AO, and PI on prefixed cell monolayers.
• Observed distinct metachromatic effects with double and triple dye combinations.
• Validated a sequential staining method for differentiating cell viability states in fixed cells.
• Showcased the utility of a long-pass FITC filter for this analysis.

Conclusions:

• HO, AO, and PI can be effectively used for cell viability assessment on prefixed samples.
• The proposed sequential staining method offers an easy and reliable approach for analyzing fixed cells.
• This technique expands the utility of these fluorescent dyes in fluorescence viability detection.