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

Assays for phagocytosis

Assays for ligand internalization

Random amine reactive labeling

Site-specific antibody labeling

Antibody internalization screening tools

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

Assays for phagocytosis

Assays for ligand Internalization

Assays for endocytosis

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

Assays for phagocytosis

Assays for ligand internalization

Assays for endocytosis

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

Assays for phagocytosis

Assays for ligand internalization

Random amine reactive labeling

Site-specific antibody labeling

Antibody internalization screening tools

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

Assays for phagocytosis

Assays for ligand Internalization

Assays for endocytosis

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

Assays for phagocytosis

Assays for ligand internalization

Assays for endocytosis

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

Scientific Poster

 

See the poster from ASCB 2013™, the Annual Meeting of American Society for Cell Biology.