pHrodo Indicators for pH Determination

Proprietary, pH-sensitive Invitrogen pHrodo dyes show little to no fluorescent signal at neutral pH and fluoresce brightly in acidic environments, making them ideal for use as pH indicators for a variety of applications. With green, red, and deep red 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:

Intracellular pH measured in murine J7741.A macrophages labeled with pHrodo Green and Red

Measuring intracellular pH using pHrodo indicators. Murine J774A.1 macrophages were labeled with Invitrogen pHrodo Green AM Intracellular pH Indicator and Invitrogen pHrodo Red 10,000 MW Dextran. The pHi was clamped to the indicated values using the Intracellular pH Calibration Buffer Kit.
 

Assays for cytosolic pH

Catalog # Name Size Price (USD) Qty
P35373 pHrodo™ Green AM Intracellular pH Indicator, 50 μL Each
595.65

Online Exclusive

614.00
Save 18.35 (3%)
P35372 pHrodo™ Red AM Intracellular pH Indicator, 50 μL Each
598.65

Online Exclusive

620.00
Save 21.35 (3%)
P35380 pHrodo™ AM Variety Pack Each
284.00
P35379 Intracellular pH Calibration Buffer Kit Each
313.65

Online Exclusive

327.00
Save 13.35 (4%)

Assays for phagocytosis

Catalog # Name Size Price (USD) Qty
P35364 pHrodo™ Red Zymosan BioParticles™ Conjugate for Phagocytosis Each
526.65

Online Exclusive

552.00
Save 25.35 (5%)
P35365 pHrodo™ Green Zymosan BioParticles™ Conjugate for Phagocytosis, 5 x 1 mg Each
532.65

Online Exclusive

552.00
Save 19.35 (4%)
P35361 pHrodo™ Red E. coli BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
540.65

Online Exclusive

560.00
Save 19.35 (3%)
P35366 pHrodo™ Green E. coli BioParticles™ Conjugate for Phagocytosis Each
534.65

Online Exclusive

550.00
Save 15.35 (3%)
A10010 pHrodo™ Red S. aureus BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
534.65

Online Exclusive

554.00
Save 19.35 (3%)
P35367 pHrodo™ Green S. aureus BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
527.65

Online Exclusive

552.00
Save 24.35 (4%)
P35360 pHrodo™ Deep Red E. coli BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
590.65

Online Exclusive

612.00
Save 21.35 (3%)
P35357 pHrodo™ Deep Red Mammalian and Bacterial Cell Labeling Kit Each
641.65

Online Exclusive

668.00
Save 26.35 (4%)

Assays for ligand internalization

Catalog # Name Size Price (USD) Qty
P10361 pHrodo™ Red Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
553.65

Online Exclusive

574.00
Save 20.35 (4%)
P35368 pHrodo™ Green Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
536.65

Online Exclusive

556.00
Save 19.35 (3%)
P35374 pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
550.65

Online Exclusive

578.00
Save 27.35 (5%)
P35375 pHrodo™ Green Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
548.65

Online Exclusive

576.00
Save 27.35 (5%)
P35376 pHrodo™ Red Transferrin Conjugate, 1 mg Each
611.65

Online Exclusive

642.00
Save 30.35 (5%)

Random amine reactive labeling

Site-specific antibody labeling

Antibody internalization screening tools

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    Time-lapse images showing activation of fluorescent dye in acidic cell environment

    Time-lapse images showing internalization, acidification, and fluorescence activation of the pHrodo Deep Red E. coli BioParticles during phagocytosis. RAW macrophage cells were plated at 10,000 cells per well one day before assay in complete medium. The following day, pHrodo Deep Red E. coli BioParticles Conjugate for Phagocytosis were added 1:10 from a 1 mg/mL stock for a probe concentration of 100 ug/mL. Cultures were set into the Onstage Incubator live cell imaging unit onboard an EVOS M7000. Over time, more pHrodo Deep Red fluorescence signal was detected, indicating more E. coli were internalized.

    Assays for cytosolic pH

    Catalog # Name Size Price (USD) Qty
    P35373 pHrodo™ Green AM Intracellular pH Indicator, 50 μL Each
    595.65

    Online Exclusive

    614.00
    Save 18.35 (3%)
    P35372 pHrodo™ Red AM Intracellular pH Indicator, 50 μL Each
    598.65

    Online Exclusive

    620.00
    Save 21.35 (3%)
    P35380 pHrodo™ AM Variety Pack Each
    284.00
    P35379 Intracellular pH Calibration Buffer Kit Each
    313.65

    Online Exclusive

    327.00
    Save 13.35 (4%)

    Assays for phagocytosis

    Assays for ligand Internalization

    Catalog # Name Size Price (USD) Qty
    P10361 pHrodo™ Red Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
    553.65

    Online Exclusive

    574.00
    Save 20.35 (4%)
    P35368 pHrodo™ Green Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
    536.65

    Online Exclusive

    556.00
    Save 19.35 (3%)

    Assays for endocytosis

    Catalog # Name Size Price (USD) Qty
    P35374 pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
    550.65

    Online Exclusive

    578.00
    Save 27.35 (5%)
    P35375 pHrodo™ Green Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
    548.65

    Online Exclusive

    576.00
    Save 27.35 (5%)
    P35376 pHrodo™ Red Transferrin Conjugate, 1 mg Each
    611.65

    Online Exclusive

    642.00
    Save 30.35 (5%)

    Random amine reactive labeling

    Site-specific antibody labeling

    Antibody internalization screening tools

    pHrodo dyes in flow cytometry

    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).

    Assays for cytosolic pH

    Catalog # Name Size Price (USD) Qty
    P35373 pHrodo™ Green AM Intracellular pH Indicator, 50 μL Each
    595.65

    Online Exclusive

    614.00
    Save 18.35 (3%)
    P35372 pHrodo™ Red AM Intracellular pH Indicator, 50 μL Each
    598.65

    Online Exclusive

    620.00
    Save 21.35 (3%)
    P35380 pHrodo™ AM Variety Pack Each
    284.00
    P35379 Intracellular pH Calibration Buffer Kit Each
    313.65

    Online Exclusive

    327.00
    Save 13.35 (4%)

    Assays for phagocytosis

    Catalog # Name Size Price (USD) Qty
    P35364 pHrodo™ Red Zymosan BioParticles™ Conjugate for Phagocytosis Each
    526.65

    Online Exclusive

    552.00
    Save 25.35 (5%)
    P35365 pHrodo™ Green Zymosan BioParticles™ Conjugate for Phagocytosis, 5 x 1 mg Each
    532.65

    Online Exclusive

    552.00
    Save 19.35 (4%)
    P35361 pHrodo™ Red E. coli BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
    540.65

    Online Exclusive

    560.00
    Save 19.35 (3%)
    P35366 pHrodo™ Green E. coli BioParticles™ Conjugate for Phagocytosis Each
    534.65

    Online Exclusive

    550.00
    Save 15.35 (3%)
    A10010 pHrodo™ Red S. aureus BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
    534.65

    Online Exclusive

    554.00
    Save 19.35 (3%)
    P35367 pHrodo™ Green S. aureus BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
    527.65

    Online Exclusive

    552.00
    Save 24.35 (4%)
    P35360 pHrodo™ Deep Red E. coli BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
    590.65

    Online Exclusive

    612.00
    Save 21.35 (3%)
    P35357 pHrodo™ Deep Red Mammalian and Bacterial Cell Labeling Kit Each
    641.65

    Online Exclusive

    668.00
    Save 26.35 (4%)

    Assays for ligand internalization

    Catalog # Name Size Price (USD) Qty
    P10361 pHrodo™ Red Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
    553.65

    Online Exclusive

    574.00
    Save 20.35 (4%)
    P35368 pHrodo™ Green Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
    536.65

    Online Exclusive

    556.00
    Save 19.35 (3%)

    Assays for endocytosis

    Catalog # Name Size Price (USD) Qty
    P35374 pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
    550.65

    Online Exclusive

    578.00
    Save 27.35 (5%)
    P35375 pHrodo™ Green Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
    548.65

    Online Exclusive

    576.00
    Save 27.35 (5%)
    P35376 pHrodo™ Red Transferrin Conjugate, 1 mg Each
    611.65

    Online Exclusive

    642.00
    Save 30.35 (5%)

    Random amine reactive labeling

    Site-specific antibody labeling

    Antibody internalization screening tools

    Dose-response

    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.

    Assays for cytosolic pH

    Catalog # Name Size Price (USD) Qty
    P35373 pHrodo™ Green AM Intracellular pH Indicator, 50 μL Each
    595.65

    Online Exclusive

    614.00
    Save 18.35 (3%)
    P35372 pHrodo™ Red AM Intracellular pH Indicator, 50 μL Each
    598.65

    Online Exclusive

    620.00
    Save 21.35 (3%)
    P35380 pHrodo™ AM Variety Pack Each
    284.00
    P35379 Intracellular pH Calibration Buffer Kit Each
    313.65

    Online Exclusive

    327.00
    Save 13.35 (4%)

    Assays for phagocytosis

    Catalog # Name Size Price (USD) Qty
    P35364 pHrodo™ Red Zymosan BioParticles™ Conjugate for Phagocytosis Each
    526.65

    Online Exclusive

    552.00
    Save 25.35 (5%)
    P35365 pHrodo™ Green Zymosan BioParticles™ Conjugate for Phagocytosis, 5 x 1 mg Each
    532.65

    Online Exclusive

    552.00
    Save 19.35 (4%)
    P35361 pHrodo™ Red E. coli BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
    540.65

    Online Exclusive

    560.00
    Save 19.35 (3%)
    P35366 pHrodo™ Green E. coli BioParticles™ Conjugate for Phagocytosis Each
    534.65

    Online Exclusive

    550.00
    Save 15.35 (3%)
    A10010 pHrodo™ Red S. aureus BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
    534.65

    Online Exclusive

    554.00
    Save 19.35 (3%)
    P35367 pHrodo™ Green S. aureus BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
    527.65

    Online Exclusive

    552.00
    Save 24.35 (4%)
    P35360 pHrodo™ Deep Red E. coli BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
    590.65

    Online Exclusive

    612.00
    Save 21.35 (3%)
    P35357 pHrodo™ Deep Red Mammalian and Bacterial Cell Labeling Kit Each
    641.65

    Online Exclusive

    668.00
    Save 26.35 (4%)

    Assays for ligand internalization

    Catalog # Name Size Price (USD) Qty
    P10361 pHrodo™ Red Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
    553.65

    Online Exclusive

    574.00
    Save 20.35 (4%)
    P35368 pHrodo™ Green Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
    536.65

    Online Exclusive

    556.00
    Save 19.35 (3%)
    P35374 pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
    550.65

    Online Exclusive

    578.00
    Save 27.35 (5%)
    P35375 pHrodo™ Green Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
    548.65

    Online Exclusive

    576.00
    Save 27.35 (5%)
    P35376 pHrodo™ Red Transferrin Conjugate, 1 mg Each
    611.65

    Online Exclusive

    642.00
    Save 30.35 (5%)

    Random amine reactive labeling

    Site-specific antibody labeling

    Antibody internalization screening tools

    Video

    Video Player is loading.
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    1x
      • Chapters
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      Time-lapse images showing activation of fluorescent dye in acidic cell environment

      Time-lapse images showing internalization, acidification, and fluorescence activation of the pHrodo Deep Red E. coli BioParticles during phagocytosis. RAW macrophage cells were plated at 10,000 cells per well one day before assay in complete medium. The following day, pHrodo Deep Red E. coli BioParticles Conjugate for Phagocytosis were added 1:10 from a 1 mg/mL stock for a probe concentration of 100 ug/mL. Cultures were set into the Onstage Incubator live cell imaging unit onboard an EVOS M7000. Over time, more pHrodo Deep Red fluorescence signal was detected, indicating more E. coli were internalized.

      Assays for cytosolic pH

      Catalog # Name Size Price (USD) Qty
      P35373 pHrodo™ Green AM Intracellular pH Indicator, 50 μL Each
      595.65

      Online Exclusive

      614.00
      Save 18.35 (3%)
      P35372 pHrodo™ Red AM Intracellular pH Indicator, 50 μL Each
      598.65

      Online Exclusive

      620.00
      Save 21.35 (3%)
      P35380 pHrodo™ AM Variety Pack Each
      284.00
      P35379 Intracellular pH Calibration Buffer Kit Each
      313.65

      Online Exclusive

      327.00
      Save 13.35 (4%)

      Assays for phagocytosis

      Assays for ligand Internalization

      Catalog # Name Size Price (USD) Qty
      P10361 pHrodo™ Red Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
      553.65

      Online Exclusive

      574.00
      Save 20.35 (4%)
      P35368 pHrodo™ Green Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
      536.65

      Online Exclusive

      556.00
      Save 19.35 (3%)

      Assays for endocytosis

      Catalog # Name Size Price (USD) Qty
      P35374 pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
      550.65

      Online Exclusive

      578.00
      Save 27.35 (5%)
      P35375 pHrodo™ Green Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
      548.65

      Online Exclusive

      576.00
      Save 27.35 (5%)
      P35376 pHrodo™ Red Transferrin Conjugate, 1 mg Each
      611.65

      Online Exclusive

      642.00
      Save 30.35 (5%)

      Random amine reactive labeling

      Site-specific antibody labeling

      Antibody internalization screening tools

      pHrodo dyes in flow cytometry

      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).

      Assays for cytosolic pH

      Catalog # Name Size Price (USD) Qty
      P35373 pHrodo™ Green AM Intracellular pH Indicator, 50 μL Each
      595.65

      Online Exclusive

      614.00
      Save 18.35 (3%)
      P35372 pHrodo™ Red AM Intracellular pH Indicator, 50 μL Each
      598.65

      Online Exclusive

      620.00
      Save 21.35 (3%)
      P35380 pHrodo™ AM Variety Pack Each
      284.00
      P35379 Intracellular pH Calibration Buffer Kit Each
      313.65

      Online Exclusive

      327.00
      Save 13.35 (4%)

      Assays for phagocytosis

      Catalog # Name Size Price (USD) Qty
      P35364 pHrodo™ Red Zymosan BioParticles™ Conjugate for Phagocytosis Each
      526.65

      Online Exclusive

      552.00
      Save 25.35 (5%)
      P35365 pHrodo™ Green Zymosan BioParticles™ Conjugate for Phagocytosis, 5 x 1 mg Each
      532.65

      Online Exclusive

      552.00
      Save 19.35 (4%)
      P35361 pHrodo™ Red E. coli BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
      540.65

      Online Exclusive

      560.00
      Save 19.35 (3%)
      P35366 pHrodo™ Green E. coli BioParticles™ Conjugate for Phagocytosis Each
      534.65

      Online Exclusive

      550.00
      Save 15.35 (3%)
      A10010 pHrodo™ Red S. aureus BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
      534.65

      Online Exclusive

      554.00
      Save 19.35 (3%)
      P35367 pHrodo™ Green S. aureus BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
      527.65

      Online Exclusive

      552.00
      Save 24.35 (4%)
      P35360 pHrodo™ Deep Red E. coli BioParticles™ Conjugate for Phagocytosis, 5 x 2 mg Each
      590.65

      Online Exclusive

      612.00
      Save 21.35 (3%)
      P35357 pHrodo™ Deep Red Mammalian and Bacterial Cell Labeling Kit Each
      641.65

      Online Exclusive

      668.00
      Save 26.35 (4%)

      Assays for ligand internalization

      Catalog # Name Size Price (USD) Qty
      P10361 pHrodo™ Red Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
      553.65

      Online Exclusive

      574.00
      Save 20.35 (4%)
      P35368 pHrodo™ Green Dextran, 10,000 MW, for Endocytosis, 0.5 mg Each
      536.65

      Online Exclusive

      556.00
      Save 19.35 (3%)

      Assays for endocytosis

      Catalog # Name Size Price (USD) Qty
      P35374 pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
      550.65

      Online Exclusive

      578.00
      Save 27.35 (5%)
      P35375 pHrodo™ Green Epidermal Growth Factor (EGF) Conjugate, 20 μg Each
      548.65

      Online Exclusive

      576.00
      Save 27.35 (5%)
      P35376 pHrodo™ Red Transferrin Conjugate, 1 mg Each
      611.65

      Online Exclusive

      642.00
      Save 30.35 (5%)

      Random amine reactive labeling

      Site-specific antibody labeling

      Antibody internalization screening tools

      Dose-response

      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. 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

      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.

      Resources

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