Fluorometric Na⁺ Evaluation in Single Cells Using Flow Cytometry: Comparison with Flame Emission Assay.

Area of Science:

• Cellular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Intracellular sodium concentration is crucial for fundamental cell functions.
• Fluorescent probes like Asante Natrium Green-2 (ANG) are vital for monitoring intracellular sodium in living cells.
• Current calibration methods for fluorescent sodium probes in situ using ionophores can lead to inaccurate results.

Purpose of the Study:

• To compare intracellular sodium concentration measurements using the ANG fluorescent probe and flame emission photometry.
• To investigate the impact of ionophores on ANG fluorescence and its correlation with actual intracellular sodium levels.
• To determine the reliability of ANG-2 for quantitative intracellular sodium assessment.

Main Methods:

• Intracellular sodium concentration in U937 cells was measured using flow cytometry with the ANG probe.
• Flame emission photometry combined with cellular water determination was used as a reference method.
• Intracellular sodium levels were manipulated using ionophores, ouabain (sodium pump inhibitor), or staurosporine (apoptosis inducer).

Main Results:

• Both ANG probe and flame photometry methods showed comparable results when sodium levels were altered by ouabain or staurosporine.
• ANG fluorescence was approximately two times lower in cells treated with ionophores compared to those with similar sodium concentrations but without ionophores.
• A significant discrepancy exists in ANG fluorescence when ionophores are used during calibration, impacting quantitative sodium assessment.

Conclusions:

• The sodium-sensitive dye ANG-2 is effective for detecting changes in intracellular sodium content and concentration in single cells and microparticles.
• ANG fluorescence, when calibrated in the presence of ionophores, does not accurately reflect the true intracellular sodium concentration.
• The observed reduction in ANG fluorescence in the presence of ionophores requires consideration for accurate quantitative intracellular sodium analysis.