pHrodo™ Indicators for Phagocytosis, Endocytosis, and Internalization
 | Molecular Probes® proprietary pH-sensitive pHrodo™ dyes serve as specific sensors of phagocytosis and endocytosis. The dyes are almost non-fluorescent at neutral pH and fluoresce brightly in acidic environments. This increase in fluorescence signal at low pH makes it ideal for studying phagocytosis and 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—making pHrodo™ phagocytosis indicators valuable for in vivo studies. With both red and green pHrodo™ dyes available you have more multiplexing opportunities as well as faster staining and more accurate results. |
pHrodo™ dyes allow you to:
- Specifically detect phagocytosis and endocytosis with a fluorogenic dye
- Reduce signal variability and improve timing in sensitive experiments
- Combine with red or green dyes for multiplexed experiments
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 1). 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.
 | Figure 1. Low extracellular fluorescence using pHrodo™ red- , or pHrodo™ Green-labeled Zymosan. pHrodo™ Red Zymosan Bioparticles® (left) or pHrodo™ Green Zymosan Bioparticles® (right) were resuspended at 2X working concentration at 1 mg/mL, and added to already plated MMM cells, then plates moved back to 37°C for 90 minutes incubation to allow phagocytosis to run to completion. NucBlue® Live ReadyProbes™ Reagent was used as DNA counter stain. These images demonstrate the Zymosan particles, those are non-fluorescent or only labeled with NucBlue® outside of the cells. When these particles are engulfed by the cells they demonstrate the bright intense signal. |
Imaging Phagocytosis and Endocytosis Assays
pHrodo™ BioParticles® Conjugates
The ready-made pHrodo™ E. coli and S. aureus BioParticles® conjugates can also be used directly in flow cytometry-based phagocytosis assays.
pHrodo™ Dextran Conjugates
The ready-made pHrodo™ dextran conjugates can be used directly in microplate-based fluorescent endocytosis assays.
| Indicator | Size | Cat. No. |
|---|---|---|
| pHrodo™ Red dextran, 10,000 MW for endocytosis | 0.5 mg | P10361 |
| pHrodo™ Green dextran, 10,000 MW for endocytosis | 0.5 mg | P35368 |
pHrodo™ Reactive Dyes
Reactive pHrodo™ dyes allow you to label any microorganism, antibody or protein of interest via thiol or amine conjugation. With the ability to label your own protein you can also use the pHrodo™ dye to attach to specific receptor ligands and study receptor internalization.
 | Figure 2. 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. |
Flow Cytometry Phagocytosis Assays
Rapid Assessment of Phagocytosis In Whole Blood Samples
We offer 2 kits that are specifically designed for rapid and assessment of phagocytosis in whole blood samples by flow cytometry. Both kits come with everything you need and sufficient reagents for approximately 100 assays.
| Indicator | Size | Cat. No. |
|---|---|---|
| pHrodo™ Red, E. coli BioParticles® Phagocytosis Kit | 1 Kit | A10025 |
pHrodo™ BioParticles® Conjugates
The ready-made pHrodo™ E. coli and S. aureus BioParticles® conjugates can also be used directly in flow cytometry-based phagocytosis assays.
pHrodo™ Reactive Dyes
Reactive pHrodo™ dyes allow you to label any microorganism, antibody or protein of interest via thiol or amine conjugation. With the ability to label your own protein you can also use the pHrodo™ dye to attach to specific receptor ligands and study receptor internalization.
Figure 3. 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. (Left) Granulocytes were gated using forward and side scatter. (Right) 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).
Microplate Phagocytosis and Endocytosis Assays
pHrodo™ BioParticles® Conjugates
The ready-made pHrodo™ E. coli and S. aureus BioParticles® conjugates can also be used directly in flow cytometry-based phagocytosis assays.
pHrodo™ Dextran Conjugates
The ready-made pHrodo™ dextran conjugates can be used directly in microplate-based fluorescent endocytosis assays.
| Indicator | Size | Cat. No. |
|---|---|---|
| pHrodo™ Red dextran, 10,000 MW for endocytosis | 0.5 mg | P10361 |
| pHrodo™ Green dextran, 10,000 MW for endocytosis | 0.5 mg | P35368 |
pHrodo™ Reactive Dyes
Reactive pHrodo™ dyes allow you to label any microorganism, antibody or protein of interest via thiol or amine conjugation. With the ability to label your own protein you can also use the pHrodo™ dye to attach to specific receptor ligands and study receptor internalization.
Figure 4. Dose-response of the phagocytosis inhibitor cytochalasin D in RAW cells using pHrodo™ Red Zymosan Bioparticles®, and effect of fixation. RAW macrophage cells were plated in complete medium and left overnight in cell culture. The following day, cells were rinsed 1X with Live Cell Imaging Solution and replaced with 20 uL of Live Cell Imaging Solution containing an eight point dose-response of the phagocytosis inhibitor cytochalasin D ranging from 10 uM to 3 pM and incubated for 15 minutes at 37°C in triplicate rows. Bioparticles were resuspended at 2X working concentration in Live Cell Imaging Solution at 2 mg/mL for E. coli and staph, or 1 mg/mL for zymosan, and added to the cells, then plates moved back to 37°C for 90 minutes incubation to allow phagocytosis to run to completion. The plates were read on a Molecular Devices FlexStation microplate reader with 490Ex/525Em, 515 cutoff for pHrodo Green particles, and 560Ex/600Em, 590 cutoff for pHrodo Red particles. Fixation was carried out by adding an equal volume of 8% PFA for a final concentration of 4% paraformaldehyde.
What Are pHrodo™ Indicators?
pHrodo™ dye is a novel, fluorogenic dye that dramatically increases in fluorescence as the pH of its surroundings becomes more acidic (Figure 5).
The optimal absorption and fluorescence emission maxima of the pHrodo™ Green dye and its conjugates are approximately 509 nm and 533 nm, respectively, while pHrodo™ Red excites at 560 nm and emits at 585 nm. Both pHrodo™ Green and pHrodo™ Red can also be excited with the 488 nm argon-ion laser installed on most flow cytometers, microscopes, and microplate readers.
 | Figure 5. pHrodo™ red (left) and pHrodo™ Green (right) 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 6). 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 (1983) 60:115–24; J Immunol Methods (1993) 162:1-7).
Figure 6. Tracking the internalization of biomolecules using pHrodo™ Green and pHrodo™ Red indicators.
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