Intracellular pH is generally between ~6.8 and 7.4 in the cytosol and ~4.5 and 6.0 in the cell’s acidic organelles. Unlike intracellular free Ca2+ concentrations, which can rapidly change by perhaps 100-fold, pH inside a cell varies by only fractions of a pH unit, and such changes may occur quite slowly.

Life Technologies offers a range of indicators for tracking intracellular pH in the cytosol or in particular organelles.  Dye conjugates are available for organelle targeting and reactive dyes are also offered for constructing unique fluorescent probes for specific applications.

Select pH indicators

Indicators for Tracking Cytosolic pH

  • Red and green dyes for multiplexing
  • Choice of ratiometric or intensity-based measurement

Sensitive pH determinations can be made in a physiological range using either fluorescent intensity or ratiometric measurements. BCECF and its AM ester can be used to make ratiometric measurements within a narrow pH range, while pHrodo™ dyes provide signal intensity modulation from pH 2 to pH 9 and with a choice of fluorescent wavelengths.

 

U2OS cells labeled with pHrodo™ Green AM Intracellular pH Indicator U2OS cells incubated with 10 µM of pHrodo™ Green AM Intracellular pH Indicator for 30 minutes at room temperature. Images were captured using a confocal microscope.

Calibration & Measurement

The pHrodo™ AM variety pack provides a choice of red or green indicators for pH measurement. When used together with the Intracellular pH Calibration Buffer Kit, the pHrodo™ dyes provide a complete solution for no-wash pH determination in live cells.

Intracellular pH determinarion using BCECF (left) and pHrodo® Red (right) dye. pHrodo™ dyes are essentially non-fluorescent at neutral pH and extremely photostable, enabling reproducible measurements. They provide signal intensity modulation from pH 2 to pH 9 with a choice of fluorescent wavelengths.

Tracking pH in Vesicles

Tracking internalization of fluorescent dextran is a routine method for analyzing pH changes in cellular compartments. Dextran conjugates of pHrodo™ dyes provide the most complete solution by allowing discrimination of vesicles from early endosomes to lysosomes,  with no quench or wash required.

pHrodo™ dyes are essentially non-fluorescent at neutral pH and provide signal intensity modulation from pH 2 to pH 9 with a choice of fluorescent wavelengths. By conjugating pHrodo™ Red together with Rhodamine Green™ onto a 10,000 MW dextran, the dual-labeled probe also provides a reference signal for tracking.

 

Using pH change to monitor phagocytosis in MMM cells using pHrodo™ Green E. coli BioParticles® Conjugate.

pH Indicators Selection Guide

  pHrodo™ Green AM Intracellular pH indicator pHrodo™ red AM Intracellular pH indicator 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF, AM)
Readout pHrodo dyes are non-fluorescent at neutral pH and increase in fluorescence emission intensity with acidification pH-dependent ratio of emission intensity (detected at 535 nm) when the dye is excited at ~490 nm versus the emission intensity when excited at ~440
Range Fluorescence increases from pH 8 to pH 4 pH range from 6.2 to 9.5
Standard filter set
FITC
TRITC Requires excitation ratio to quantify pH
Fluorophore pHrodo™ Green pHrodo™ Red BCECF
Ex/Em (mn) 509/533 560/585 ~440-490/535
Cell permeant or impermeant? Permeant Permeant Permeant
Usage Notes Load in cell media
Platforms Flow cytometer, microplate reader, imaging Microplate reader
Format 50 µL of 5 mM (1,000x) 50 µL of 5 mM (1,000x) 20 x 50 µg
Cat. No. P35373 P35372 B1170
  pHrodo™ (AM) Variety pack Intracellular pH Calibration Kit
Readout pHrodo™ dyes are non-fluorescent at neutral pH and increase in fluorescence emission intensity with acidification Measure exact pH in live cells with pHrodo™ red AM and pHrodo™ green AM
Range Fluorescence increases from pH 8 to pH 4 pH range from 4.5 to 7.5
Standard filter set FITC TRITC
Valinomycin and nigericin to create membrane pores
Fluorophore pHrodo™ Green pHrodo™ Red
Ex/Em (mn) 509/533 560/585
Cell permeant or impermeant? Permeant Permeant Permeabilizes cell membrane
Usage Notes Load in cell media Cellular pH equilibrates with medium
Platforms Flow cytometer, microplate reader, imaging
Format 1 kit 1 kit
Cat. No. P35380 P35379
  pHrodo™ Green Dextran, 10,000 MW pHrodo™ Red Dextran, 10,000 MW Dextran, BCECF, 10,000 MW, Anionic
Readout pHrodo™ dyes are non-fluorescent at neutral pH and increase in fluorescence emission intensity with acidification pH-dependent ratio of emission intensity (detected at 535 nm) when the dye is excited at ~490 nm versus the emission intensity when excited at ~440
Range Fluorescence increases from pH 8 to pH 4 pH range from 6.2 to 9.5
Vehicle Dextran 10,000 MW Dextran 10,000 MW Dextran 10,000 MW
Standard filter set FITC TRITC Requires excitation ratio to quantify pH
Fluorophore pHrodo™ Green pHrodo™ Red BCECF
Ex/Em (nm) 509/533 560/585 ~440-490/535
Cell permeant or impermeant? Impermeant Impermeant Impermeant
Usage notes Loaded by endocytosis, microinjection, patch pipette of pinocytic loading agent Loaded by endocytosis, microinjection, patch pipette, or pinocytic loading agent
Platforms Flow cytometer, microplate, Imaging Imaging
Format 0.5 mg 0.5 mg 10 mg
Cat. No. P35368 P10361 D1878

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. 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.

 

 

pHrodo™ red and pHrodo™ Green 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.

 

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.