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Proprietary, pH-sensitive Molecular Probes® pHrodo™ dyes are almost non-fluorescent at neutral pH and fluoresce brightly in acidic environments, making them ideal for use as pH indicators for a variety of applications. With both red and green pHrodo™ dye choices, you have more opportunities for multiplexing pH determination with other cellular measurements.
pHrodo™ dyes can be used to measure intracellular pH in either the cytosol or in cellular compartments. The minimal dye fluorescence at neutral pH also eliminates the need for wash steps and quencher dyes because any non-internalized dye is essentially nonfluorescent in the culture medium.
pHrodo™ dyes allow you to:
- Specifically detect and monitor phagocytosis and endocytosis with pHrodo™ dyes
- Measure cytosolic pH using pHrodo™ dyes, and calibrate with pH standards
- Track antibody internalization with pHrodo™ conjugates
- Track ligand internalization with pHrodo™ conjugates
- Combine with red or green dyes for multiplexed experiments
Measuring intracellular pH using pHrodo™ indicators
Murine J774A.1 macrophages were labeled with pHrodo™ Green AM Intracellular pH Indicator and pHrodo™ Red 10,000 MW Dextran. The pHi was clamped to the indicated values using the Intracellular pH Calibration Buffer Kit.
 | 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. |
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).
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.
| 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. |
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).
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.
 | 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. |
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