pHrodo™ Dyes for Amine Labeling

Perform cell internalization studies with low signal-to-background and high specificity using the pHrodo pH sensor dyes, including pHrodo Red, Green, and Deep Red dyes. These dyes can be conjugated to biomolecules such as antibodies, and become highly fluorescent only when found in acidic environments, such as those of cell lysosomes and endosomes. This enables detection of the conjugated biomolecule in the live cell assay without the need for wash steps or quencher dyes in the experimental workflow.

pHrodo Red, Green, and Deep Red dyes
The amine-reactive pH-sensitive pHrodo Red, Green, and Deep Red dyes are optimized for the creation of bioconjugates used in the study of antibody internalization, endocytosis, and phagocytosis in live cell assays. Amine-reactive pHrodo dyes enable you to label primary amines on an antibody, protein, cell, virus, or other amine-containing biomolecule to create a conjugate that fluoresces inside acidic environments but shows little to no signal at neutral pH. The lack of fluorescence of pHrodo dyes in a typical extracellular environment eliminates the need for wash steps or quencher dyes in the experimental workflow.

pHrodo iFL Red and Green STP Ester dyes
pHrodo iFL Red and Green STP Ester dyes are improved versions of the original pHrodo Red and Green dyes and are optimized to create more soluble conjugates than the original dyes. This enhanced property makes them useful for the labeling of antibodies that may otherwise precipitate out of solution during conjugation.

pHrodo Deep Red TFP Ester
The pHrodo Deep Red TFP Ester is distinct from pHrodo iFL Red and Green Ester dyes in that it has a lower pKa and, as a result, shows both improved signal-to-background ratios and fluoresces later in the endocytic pathway. Our low-background pHrodo Deep Red dye makes it easier than ever to study internalization with a higher certainty of results and less optimization because pHrodo Deep Red dye only fluoresces in the late endosome and lysosome.

Features and benefits of the pHrodo dyes for amine labeling

• Customization—use pH-sensitive pHrodo dyes to make pH-sensitive bioconjugates of your choice
• Speed and accuracy—get faster and more accurate results than with any other endocytosis or phagocytosis assay, no need for wash steps or quenchers
• Multiplexing—multiplex with green or red fluorescent markers such as GFP or RFP with three different color choices
• Superior signal window—with pHrodo Deep Red dye’s minimal background at neutral pH, maximal signal-to-noise ratio is enabled for certainty of results
• Low pKa—pHrodo Deep Red dye eliminates non-specific signal from non-internalized cargo, resulting in fluorescence only in late endosomes and lysosomes

The increase in fluorescence of pHrodo dyes as the pH acidifies correlates with the acidification of intracellular vesicles, making these dyes valuable tools for the study of endocytosis or phagocytosis and their regulation by environmental factors, drugs, or pathogens. The spectral properties of pHrodo dyes also make them useful for multi-color experiments. pHrodo iFl Red, iFL Green, and Deep Red dyes can be detected using most standard red, green, and deep red fluorescent filter sets. pHrodo dye conjugates have been validated for use in a variety of applications, including flow cytometry, fluorescent microscopy, and high content screening (HCS).

pHrodo Deep Red dye has a lower pKa by design and shows no signal in neutral conditions, fluorescing only in acidic environments. This unique property enables rapid assay development and certainty of results when investigating antibody internalization and endocytic/phagocytic pathways. pHrodo Deep Red dye enables better discrimination of internalized cargo from outside the cell, whereas pHrodo iFl Red and Green dyes fluoresce in the early endosome.