Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis

Area of Science:

• Immunophenotyping
• Flow Cytometry
• Cell Viability Assays

Background:

• Propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) viability stains overlap spectrally with RPE-Cy5 when excited at 488 nm.
• This spectral overlap limits their use in three-color immunophenotyping, especially for low-level marker expression.
• Hydroxystilbamidine methanesulfonate (FG), an ultraviolet-excitable dye, binds DNA in dead or permeabilized cells.

Purpose of the Study:

• To assess the suitability of hydroxystilbamidine methanesulfonate (FG) as a viability stain.
• To compare FG with propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) for detecting nonviable cells.
• To evaluate FG's compatibility with common fluorochromes in multicolor analysis.

Main Methods:

• Compared FG's ability to detect nonviable cells in human peripheral blood with PI and 7-AAD.
• Assessed FG staining stability and the impact of dye/cell concentration on discrimination.
• Measured median fluorescence of viable and nonviable cells to determine discrimination.

Main Results:

• FG labeling is stable for at least 3 hours across a wide cell concentration range (4 x 10^5 to 4 x 10^7 cells/ml).
• FG staining concentrations of 2-8 microM are effective.
• Cell viability determined by FG strongly correlates with PI (r=0.9636) and 7-AAD (r=0.9879) estimates.

Conclusions:

• Hydroxystilbamidine methanesulfonate (FG) is a reliable alternative viability stain.
• FG can be used with fluorochromes such as FITC, PE, and RPE-Cy5.
• FG is suitable for multicolor analysis using dual-laser instruments.