PH Measurements With Snarf1 Using Nigericin Calibration

For most purposes, SNARF-1 is the most satisfactory pH; probe currently available. It has a clear isobestic point (i.e., a fluorescence emission wavelength that is insensitive to pH change), and it is very sensitive to pH; changes within the physiological range. Calibration is achieved by resuspending dye-loaded cells in high-potassium buffers in the presence of the proton ionophore nigericin. Nigericin allows exchange of H+ for K+ across their concentration gradients, so that when external and internal potassium ion concentrations are approximately equal, there is free movement of hydrogen ions and intracellular pH is the same as extracellular pH. This feature allows the experimenter to set pH; in a series of calibration standards to a set of known values, and thus to construct a calibration curve of fluorescence versus pH;.

Materials

Cell sample

PBS (APPENDIX 2A)

2 mM carboxy SNARF-1 AM (see recipe) High-potassium calibration buffers (see Support Protocol 1) 1 mg/ml nigericin (see recipe)

12 x 75-mm polypropylene or polystyrene tubes (Falcon) as required by flow cytometer

Flow cytometer with 488-nm argon laser and ratio parameter, if available Bandpass filters centered at or around 580 and 640 nm

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