pHrodo™ Deep Red Mammalian and Bacterial Cell Labeling Kit

The pHrodo iFL Red Microscale Protein Labeling Kit and pHrodo iFL Green Microscale Protein Labeling Kit provide the reagents needed to easily label three microscale purified protein or antibody samples (20–100 μg) with pHrodo iFL Red or pHrodo iFL Green dye. These kits contain amine-reactive pH-sensitive pHrodo iFL Red or Green STP ester dye that covalently binds to free lysines anywhere they occur in an antibody or protein.

pHrodo iFL Red and pHrodo iFL Green dyes are improved versions of pHrodo Red and pHrodo Green dyes and optimized for the creation of bright, soluble bioconjugates for use in the study of antibody internalization, endocytosis, and phagocytosis. They are also more soluble than the original pHrodo Red and pHrodo Green dyes, making them useful for the labeling of antibodies that may otherwise precipitate out of solution during conjugation. pHrodo iFL Red and pHrodo iFL Green dyes are non-fluorescent outside the cell, but fluoresce bright red or bright green as the pH of their surroundings becomes more acidic, such as those found inside of phagosomes and endosomes.

The pHrodo iFL Red and Green microscale labeling kits offer the following benefits:
• Convenience—labeling typically takes two hours, with fewer than 30 minutes of hands-on time
• Versatility—labelling can be performed on 12–150 kDa molecular weight proteins
• Customization—create your own probes for studying antibody internalization, phagocytosis, and endocytosis

The pHrodo Deep Red Mammalian and Bacterial Cell Labeling Kit provides the reagents needed to easily label live cells (mammalian or bacterial) for the study of phagocytosis and endocytosis. It is optimized for the discrimination of phagocytosed and non-phagocytosed cells because the pH-sensitive, low-background pHrodo Deep Red dye shows no signal in neutral conditions and fluoresces only when exposed to acidic environments. This unique property of pHrodo Deep Red permits rapid assay development and certainty of results when investigating endocytic and phagocytic pathways. pHrodo Deep Red dye offers better discrimination of internalized cargo from outside the cell because it has an approximate pKa of 5 and does not fluoresce until it enters the late endosome and lysosome. pHrodo Deep Red dye can be detected using a Cy5 fluorescent filter set and has been validated for use in a variety of applications, including flow cytometry, fluorescent microscopy, and high content analysis (HCA).

The pHrodo Deep Red Mammalian and Bacterial Cell Labeling Kit offers the following benefits:
• Multiplexing—pHrodo Deep Red dye can be multiplexed with a wide variety of blue, green, and red dyes and reporters such as GFP and RFPs, Mitosox Green or Red, CellEvent Caspase 3/7 Green or Red, NucBlue, or TMR, among many others
• Simplicity—easy to follow, step-by-step protocol
• Superior signal window—minimal background at neutral pH enables maximal signal-to-noise ratio for certainty of results
• Low pKa—eliminates non-specific signal from non-internalized cargo and only fluoresces in late endosomes and lysosomes