pHrodo™ Indicators—Enlightening Endocytosis
![]() | 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:
Order our pHrodo™ dye products for endocytosis and phagocytosis |
What is pHrodo™ dye?
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.
Specific Detection of Phagocytosis and Endocytosis
Reproducible Results in Less Time
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.

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.
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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. |

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).
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