Real-Time Phagocytosis Assays Made Simple
Phagocytosis, the process by which cells engulf large particles such as apoptotic cells, pathogens, or cellular debris, is essential for immunity and tissue homeostasis. While many cell types can phagocytose, macrophages are the primary professional phagocytes, clearing billions of dying cells every day.
Traditional phagocytosis assays rely on labor-intensive workflows and provide only limited endpoint measurements. In contrast, xCELLigence Real-Time Cell Analysis (RTCA) offers a faster, more informative alternative.
A Smarter Way to Measure Phagocytosis
With xCELLigence technologies, researchers gain access to a real-time, label-free, and highly sensitive phagocytosis assay that delivers:
Effortless live-cell detection of phagocytic activity through integrated imaging.
Quantitative, reproducible data with minimal manual steps—no fixing, quenching, or washing.
Sensitive, high-throughput kinetic readouts over hours to days.
Detailed insights into compounds or conditions that enhance or inhibit phagocytosis.
Quantify Microbial Killing by Phagocytosis in Real Time
Traditional phagocytosis assays are labor-intensive and offer only limited endpoint snapshots. With xCELLigence RTCA eSight, you can continuously monitor how effectively immune cells engulf and eliminate microorganisms, without complex workflows.
This simple mix-and-read assay delivers real-time kinetic data and high-quality visualization for both gram-negative and gram-positive bacteria. As bacteria are internalized and enter the acidic phagosome, their fluorescence intensifies, enabling rapid, automated, and highly sensitive detection of phagocytic activity.
Visualization of RAW 264.7 macrophages (expressing GFP) phagocytosing pHrodo Red E.coli bioparticles
Quantifying phagocytosis using pHrodo Red E. coli Bioparticles.
(A) pHrodo Red fluorescence increases sharply as pH drops from 7.4 to 4.0. (B) BioParticles remain non-fluorescent outside the cell but emit strong red fluorescence once internalized into the acidic phagolysosome.
Track Phagocytosis with pH-Sensitive Fluorescent Reagents
Monitor phagocytic activity in real time using pH-responsive fluorescent bioparticles. In this example, macrophages are cocultured with E. coli labeled with a pH-sensitive red dye. These BioParticles remain non-fluorescent at neutral pH but emit a strong red signal once internalized into the acidic phagosome.
As the fluorescence increases, the assay delivers clear, noninvasive, and quantitative readouts of phagocytosis kinetics—ideal for studying receptor–ligand interactions and evaluating pathway modulators.
Visualize and Quantify Real-Time Phagocytosis Kinetics
Track phagocytosis as it happens with xCELLigence RTCA eSight. RAW 264.7 macrophages are exposed to pHrodo Red E. coli BioParticles, enabling simultaneous live-cell imaging and kinetic analysis. By measuring total red signal area and intensity over time, eSight delivers clear, quantitative insights into the rate and progression of phagocytic activity.
Application Note: Visualization and Quantitative Analysis of Macrophage Phagocytosis
Kinetics of phagocytosis visualized with pHrodo Red E. coli BioParticles.(A) Time-lapse images show increasing red fluorescence as RAW 264.7 macrophages internalize BioParticles. (B) Quantification using total red area confirms progressive phagocytosis, with no signal detected in wells lacking macrophages. (C) Phagocytosis kinetics can be quantified using either red area or red intensity measurements.
Dose-dependent inhibition of phagocytosis by cytochalasin D.
(A) After a 2-hour coincubation, RAW 264.7 macrophages (10k/well) show progressively reduced uptake of pHrodo Red E. coli BioParticles (6.25 µg) with increasing cytochalasin D concentrations. Scale bar: 120 µm. (B) Real-time total red fluorescence area highlights the dose-dependent suppression of phagocytosis. (C) Dose-response curve generated from area-under-curve values in panel B, yielding an IC₅₀ of 11 µg/mL (n=3).
Evaluate Dose-Dependent Inhibition of Phagocytosis
Using xCELLigence RTCA eSight, RAW 264.7 macrophages can be screened for compounds that modulate phagocytosis. In this example, cytochalasin D reduces phagocytic activity in a clear dose-dependent manner. By plotting the area under the real-time curves, an IC₅₀ of 11 µg/mL is obtained—demonstrating how eSight enables fast, quantitative assessment of phagocytosis inhibitors.