pHrodo™ Indicators—Enlightening Endocytosis

pHrodo™ Indicators The new Molecular Probes® proprietary pH-sensitive pHrodo™ dye is a specific sensor of endocytosis.  The dye is nonfluorescent at neutral pH and fluoresces bright red in acidic environments. This increase in fluorescence signal at low pH makes it ideal for studying endocytosis and its regulation by drugs and/or environmental factors.  The minimal dye fluorescence at neutral pH also eliminates the need for wash steps and quencher dyes because any non-internalized dye will be essentially nonfluorescent. You’ll get faster staining and more accurate results.  The pHrodo™ dye allows you to:

  • Specifically detect phagocytosis and endocytosis with a fluorogenic dye
  • Reduce signal variability and improve timing in sensitive experiments
  • Multiplex with green dyes such as GFP, Fluo-4, or calcein

Order our pHrodo™ dye products for endocytosis and phagocytosis

What is pHrodo™ dye?

pHrodo™ dye is a novel, rhodamine-based, fluorogenic dye that dramatically increases in fluorescence as the pH of its surroundings becomes more acidic (Figure 1). The amine-reactive succinimidyl ester form of this dye (pHrodo™ SE) has a pKa of ~7.3 in solution, which shifts to about ~6.5 upon conjugation to the K-12 strain of E. coli or the protein A-free Wood strain of S. aureus used in this product line.

The optimal absorption and fluorescence emission maxima of the pHrodo™ dye and its conjugates is approximately 560 nm and 585 nm, respectively. However, the fluorophore is readily excited with the 488 nm argon-ion laser installed on most flow cytometers, microscopes and microplate readers.

pHrodo dye fluorescence emission
Figure 1. pHrodo™ dye fluorescence emission spectra. The fluorescence emission spectra of pHrodo™ dye–labeled E. coli were measured in a series of 50 mM potassium phosphate buffers ranging in pH from 4 to 10. The E. coli were at a concentration of 0.1 mg/mL, and the readings were made on a Hitachi F4500 fluorometer, using an excitation wavelength of 532 nm.

Specific Detection of Phagocytosis and Endocytosis

The unique pHrodo™-based system measures phagocytosis and endocytosis based on acidification of the particle or protein conjugates as they are ingested by cells (Figure 2). With a simple no-cell background subtraction method, a large and specific signal is obtained from cells that ingest the particles, providing a specific index of phagocytosis with a variety of pretreatments or conditions.   This system offers key advantages over the classical nonfluorogenic indicators of bacterial uptake where washing and quenching steps are required (J Immunol Methods 60, 115 (1983); J Immunol Methods 162, 1 (1993)).

pHrodo dye-based schematic of phagocytosis
Figure 2.  Schematic of pHrodo™ dye-based detection of phagocytosis. Particles (microorganisms or proteins) labeled with pHrodo™ dye are added to cells. Some remain in solution or become non-specifically attached to cell—they do not fluoresce because of the neutral pH of the extracellular environment. Some are taken up by phagocytosis and become encapsulated in vesicles. As vesicles are processed, pH decreases and the pHrodo™ -labeled particles fluoresce brightly red.

Reproducible Results in Less Time

The lack of fluorescent signal at neutral pH with the pHrodo™ dye conjugates prevents the detection of non-internalized and nonspecifically bound conjugates, eliminating the uncertainty of whether particle signals derive from phagocytosis activity or extracellular sources (Figure 3).  Without the fluorescence background, you can achieve more sensitive detection of phagocytosis and endocytosis. There is also no need for quenching reagents and extra wash steps, saving you time and allowing you to design more complex experiments.

The pHrodo™ dye can also be used in multiplexing experiments, enabling you to get more out of your experiment. The red fluorescence of the dye makes it compatible for use with green fluorescent dyes that are commonly used in cellular analysis. These include green fluorescent proteins (GFPs), fluo-4, fluorescein, Alexa Fluor® 488 and calcein.
pHrodo Dye Low Fluorescence Background
Figure 3. Low extracellular fluorescence using pHrodo™-labeled E.coli. The green  fluorescent dye, calcein, was used as a cytosolic stain to show the outline of macrophage cells. These images demonstrate that pHrodo™ labeled E.coli are relatively nonfluorescent outside the cell. Only after internalization and acidification of the phagosome is the red fluorescence clearly visible. The images also show the possibility of multiplexing the red pHrodo™ dye with green dyes such as fluorescein, Alexa Fluor® 488 conjugates, cell tracers, GFP, and Fluo-4 calcium sensor.

Flow Cytometry Phagocytosis Assays

We offer 2 kits that are specifically designed for rapid and assessment of phagocytosis in whole blood samples by flow cytometry (Figure 4). Both kits come with everything you need and sufficient reagents for approximately 100 assays.

Imaging and Microplate Phagocytosis Assays

The ready-made pHrodo™ E.coli and S.aureus BioParticles® conjugates can be used directly in fluorescence imaging (Figure 5) or in microplate  phagocytosis assays.


The amine reactive form, pHrodo™ SE, allows you to label any microorganism or protein of interest. The ability to label your own protein could also allow you to use the pHrodo™ dye to attach to specific receptor ligands and study receptor internalization.

pHrodo Dyes in Flow Cytometry
Figure 4. pHrodo™ dyes in flow cytometry. The utility of pHrodo™ labeled bacteria extends beyond plate readers and imaging platforms to flow cytometry. Here, flow cytometric analysis shows the increased fluorescence of granulocytes treated with pHrodo™ dye–labeled E. coli. A whole blood sample was collected and treated with heparin, and two 100 µl aliquots were prepared. Both aliquots were treated with pHrodo™ dye–labeled E. coli and vortexed. One sample was placed in a 37°C water bath, and the other sample (negative control) was placed in an ice bath. After red blood cells lysis and centrifugation samples were then analyzed on a FACSCalibur™ cytometer (BD Biosciences) using a 488 nm argon laser and 564-606 nm emission filter. (A) Granulocytes were gated using forward and side scatter. (B) The sample incubated at 37°C shows the increased fluorescence of the intracellular pHrodo™ dye–labeled E. coli (red), in contrast to the negative control sample, which was kept on ice to inhibit phagocytosis (blue).

Phagocytosis of pHrodo Dye Time Lapse
Figure 5. Time-lapse images showing internalization and acidification of the pHrodo™ E.coli BioParticles® conjugate during phagocytosis by Murine J774A.1 cells. Using excitation with a 561 nm laser, fluorescence images were recorded in the 570–699 nm emission range and white- light DIC images were recorded on a separate PMT. Images were collected every 30 sec for 83.8 min (not all images are shown) using a Leica TCS SP5 laser confocal microscope employing a 63× (NA 1.4) oil objective and the resonant galvanometer scanner mode (8000 Hz). Image contributed by Lucy Deriy and Deborah Nelson, University of Chicago.