Intracellular pH Measured by ADB

Abstract

This protocol is used to measure the intracellular pH (pH_i) of cells using the dye 2,3-dicyano-l,4-hydroquinone diacetate (ADB). This dye readily passes through the plasma membrane and is cleaved by cellular esterases to form free dicyanohydroquinone (DCH) in the cell. The DCH shifts its fluorescence spectrum dependent upon the intracellular pH.¹ The ratiop fluorescence spectrum dependent upon the intracellular pH_i. DCH is excited by the argon UV laser lines, which limits its usefullness to users with UV lasers. It does give high resolution pH>_i measurements with coefficients of variation (CVs) of 3–4% and can measure differences in pH_i of <0.05 pH units². A advantage is that it readily leaks out of cells, so the timing of measurements must be carefully controlled.