Fluorogenic pH sensitive dyes and their method of use

A method for detecting phagocytosis of a carrier molecule in a solution, the method comprising:

• (a) conjugating a carrier molecule to a pH-sensitive compound of Formula I
• wherein,
• R1, R2, R3 and R6 are each independently H, Z, or an electron donating group (EDG), provided that R1 and R2 are not hydroxyl or thiol or their deprotonated forms;
• R4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
• R5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
• X is a fluorophore;
• Y is ═CRbRcor ═CRbRd;
• Z is —ORc, —SRc, —NRbRc;
• Rb is H, alkyl, or substituted alkyl;
• Rc is alkyl or substituted alkyl;
• and
• Rd is amino or substituted amino;
• or a stereoisomer, tautomer, or salt thereof to form a carrier conjugate, wherein the pH-sensitive compound has a pKa value of about 6-7;
• (b) contacting the carrier conjugate with a cell to form a contacted cell;
• (c) incubating the contacted cell to form an incubated solution;
• (d) illuminating the incubated solution to form an illuminated solution;
• and
• (e) detecting fluorescent emissions from the illuminated solution;
• wherein fluorescent emissions indicate phagocytosis of the carrier molecule, and wherein no quenching step is needed before the detecting step.