pHrodo™ Red and Green AM Intracellular pH Indicator Dyes

Measure and quantify intracellular pH with pHrodo Red and Green AM indicator dyes, which are fluorogenic probes that traverse the cell membrane and remain within the intracellular space upon cleavage by nonspecific esterases. pHrodo Red and Green AM indicator dyes become increasingly fluorescent as the pH drops and can be used to quantify cytosolic pH in the range of 4–9. These indicator dyes can also be used in multiplex experiments within HCS, flow cytometry, microplate fluorometry, and HCS applications.

pHrodo Red (Cat. No. P35372) and Green (Cat. No. P35373) AM intracellular pH indicators are fluorogenic probes used to measure intracellular pH in live cells. They are weakly fluorescent at neutral pH but become increasingly fluorescent as the pH decreases. These reagents can be used to quantify cellular cytosolic pH in the range of 4–9 with a pKa of ∼6.5 and with excitation/emission spectra of 560/585 nm (pHrodo Red) or 509/533 nm (pHrodo Green).

Both pHrodo Red and pHrodo Green pH indicator dyes are photostable and appropriate for use in multiplexing experiments. pHrodo Red easily multiplexes with a wide variety of blue, green, and far red dyes and reporters such as GFP, Fuo-4, calcein, NucBlue, CellEvent Caspase 3/7 green, Mitosox Green, and Mitotracker Deep Red, among many others. pHrodo Green easily multiplexes with a wide variety of blue, red, and far red dyes reporters such as Mitosox Red, CellEvent Caspase 3/7 Red, NucBlue, RFPs, and Mitotracker Deep Red, among many others.

The pHrodo Red and Green dyes have been modified with AM ester groups, resulting in an uncharged molecule that can permeate cell membranes. Once inside the cell, the lipophilic blocking groups of the dyes are cleaved by nonspecific esterases, resulting in compounds that are retained within the intracellular space. The fluorescence intensity of the probe then becomes an indicator of intracellular pH. Subsequent use of the Intracellular pH Calibration Buffer Kit (Cat. No. P35379) allows this intracellular pH to be quantified.

pHrodo Red and Green AM intracellular pH indicator dyes are compatible with various platforms, such as traditional fluorescence microscopy, high content screening (HCS), flow cytometry, and microplate-based fluorimetry or high throughput screening (HTS). These reagents are also compatible with widefield or confocal fluorescence microscopy, flow cytometry, fluorescence plate readers, and high content instruments.