Phagosomal pH determination by dual fluorescence flow cytometry

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Accurate measurement of phagosome pH is crucial for understanding immune cell function.
• Existing methods using fluorescein derivatives and spectrofluorimetry, fluorescence microscopy, or flow cytometry have significant drawbacks, including slow or inaccurate readings.
• Phagosome maturation involves a decrease in pH, impacting the degradation of engulfed pathogens.

Observation:

• A new method was developed for phagosome pH determination using dual-labeled zymozan particles in J774 cells.
• Dual fluorescence flow cytometry with an argon-ion laser at 488 nm enabled rapid, specific measurement of phagosome fluorescence.
• Oregon Green 488, a pH-sensitive probe with a lower pKa than carboxyfluorescein, allowed pH investigation below 5.
• Dual labeling with pH-insensitive carboxytetramethylrhodamine overcame spectral limitations of Oregon Green 488.
• A calibration curve using ionophores established phagosomal pH based on fluorescence intensity ratios.

Findings:

• The developed method accurately determines phagosomal pH as 4.5 ± 0.1.
• This technique allows for pH measurements as low as 4.
• The method is effective even in the presence of non-engulfed reporter particles.

Implications:

• This rapid and accurate method offers a significant improvement over existing techniques for phagosome pH measurement.
• It provides a valuable tool for studying phagosome maturation and function in various biological contexts.
• The improved accuracy and lower pH detection limit can advance research in infectious diseases and immune responses.