Measuring Intracellular pH Using SNARF-1

Abstract

This protocol is used to measure the intracellular pH (pH_i) of cells using the dye carboxy-seminaphthorhodafluor (SNARFO. The acetoxy methyl ester form of this dye readily passes through the plasma membrane and is cleaved by cellular esterases to form free SNARF-l in the cell. SNARF-1 shifts its fluorescence spectrum dependent upon the intracellular pH. The ratio of fluorencence in two different bands is proportional to pH_i. The pK_a is about 7.5, so SNARF-1 is better measurement pH values between about 7 and 8. In contrast, DCH(see protocol) is better for more acidic pH values. SNARF-1 is excited by the argon laser lines at 488 or 514nm excitation gives a stronger signal. It gives high resolution pH_i measuerements with coefficients of variation (CVs) of 2—4% and can measure differences in pH_i of <0.05 pH units.² In contrast to DCH, it does not leak rapidly out of cells, so cells can be preloaded with SNARF-1, then treated with various agents and the pH_i measured immediately after the treatment. This makes it more flexible for measuring pH_i. We have found, however, that it consistently gives a higher value of pH_i than that obtained by DCH¹, and DCH is closer to the absolute value.