Determination of Peroxisomal pH in Living Mammalian Cells Using pHRed
Methods in Molecular Biology
Humana Press (Springer Imprint)
Reason for embargo
Organelle pH homeostasis is crucial for maintaining proper cellular function. The nature of the peroxisomal pH remains somewhat controversial, with several studies reporting conflicting results. Here, we describe in detail a rapid and accurate method for the measurement of peroxisomal pH, using the pHRed sensor protein and confocal microscopy of living mammalian cells. pHRed, a ratiometric sensor of pH, is targeted to the peroxisomes by virtue of a C-terminal targeting sequence. The probe has a maximum fluorescence emission at 610 nm while exhibiting dual excitation peaks at 440 nm and 585 nm, allowing for ratiometric imaging and determination of intracellular pH in live cell microscopy.
This work was supported by the Portuguese Foundation for Science and Technology (FCT) (SFRH/BPD/90084/2012 to L. G.).
This is the author accepted manuscript. The final version is available from Humana Press via the DOI in this record.
Vol. 1595, pp. 181-189