A rapid microplate-based fluorometric assay for phagocytosis

Area of Science:

• Immunology
• Cell Biology
• Assay Development

Background:

• Phagocytosis is a critical cellular process for immune response and cellular debris clearance.
• Traditional methods for quantifying phagocytosis, such as spectrophotometry and flow cytometry, can be time-consuming and require large sample volumes.
• There is a need for a more efficient and sensitive assay for phagocytosis measurement.

Purpose of the Study:

• To adapt a quantitative fluorometric assay for phagocytosis measurement using a 96-well fluorescence plate reader.
• To optimize the assay for high throughput analysis with reduced sample volume.
• To compare the efficacy of different plate types for enhanced sensitivity and reduced variability.

Main Methods:

• A fluorometric assay using fluorescein-conjugated polystyrene beads was adapted for a 96-well fluorescence plate reader.
• Opaque white 96-well luminostrips were compared with black fluoroplates for fluorescent output and sensitivity.
• Macrophage monolayers were incubated with beads, washed, lysed, and phagocytosis was quantified by measuring fluorescent intensity.

Main Results:

• Opaque white 96-well luminostrips demonstrated a 100-fold greater fluorescent output and higher sensitivity compared to black fluoroplates.
• White luminostrips also exhibited significantly lower intraplate variability.
• The assay successfully quantified phagocytic activity in resident and elicited peritoneal macrophages.

Conclusions:

• The adapted 96-well plate reader assay provides a highly sensitive and efficient method for quantifying phagocytosis.
• The use of opaque white luminostrips significantly enhances assay performance.
• This optimized assay is suitable for rapid analysis of large sample numbers in macrophage research.