An improved method to quantitate phagocytosis with mineral oil particles which avoid cell flotation

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Phagocytosis is a critical cellular process mediated by immune cells like polymorphonuclear leukocytes (PMN).
• Accurate quantitation of phagocytosis is essential for understanding immune function and disease.
• Previous methods using Oil Red O-stained paraffin oil particles underestimated phagocytosis due to cell flotation.

Purpose of the Study:

• To develop a reliable method for quantifying phagocytosis by human PMN.
• To overcome the underestimation issue caused by cell flotation in existing assays.
• To establish a new assay for measuring both the rate and capacity of phagocytosis.

Main Methods:

• Prepared paraffin oil-silicon oil particles with densities ranging from 0.8740 to 0.9952 g/cm³.
• Utilized particles with sufficient density to prevent PMN flotation during phagocytosis assays.
• Quantitated phagocytosis using Oil Red O dye and measured uptake by PMN.

Main Results:

• A density of 0.9952 g/cm³ for paraffin oil-silicon oil particles was found to prevent PMN flotation.
• The new assay revealed an initial phagocytosis rate of 400 µg/min/5x10⁶ PMN, approximately 20 times higher than previously reported.
• Maximum phagocytic capacity was determined to be 2.5 mg/5x10⁶ PMN, and particle uptake was sensitive to known phagocytosis inhibitors.

Conclusions:

• A novel, accurate assay for quantifying human PMN phagocytosis has been established using density-adjusted oil particles.
• This method provides significantly higher measurements of phagocytic rate and capacity compared to older techniques.
• The assay is sensitive to inhibitors and can detect alterations in phagocytosis, such as in myeloperoxidase-deficient PMN.