We have published a brief Youtube video showing a phagosomal pH measurement experiment. This method can be used to assess phagosome, efferosome or endosome acidification using any target that can be conjugated to FITC, and using any cell type. The method automatically compensates for photobleaching and incorporates an in situ pH calibration to convert FITC ratios into pH units.
In this video the pH of phagosomes in primary human macrophages following uptake of IgG-coated 5µm beads (pathogen mimics) is quantified. pH is measured by imaging FITC conjugated to the beads using two excitation wavelengths – a 440 nm excitation, which is pH-independent and allows for photobleaching correction, and a 490 nm excitation which is pH-dependent, with emission decreasing with decreasing pH.
After the time-lapse video is completed a calibration curve is calculated by profusing the imaging chamber with nigericin-containing buffer at known pH’s (4.0, 5.0, 6.0 and 7.0). The nigericin acts as a proton ionophore, equalizing the pH in the phagosome/efferosome lumen to the pH of the extracellular media. FITC images at 440 nm and 490 nm excitation are captured for each pH.
Post-imaging, the background is subtracted from the Ex440 and Ex490 channels and the 490/440 ratio calculated. A pH can be assigned to each bead based on the calibration curve generated using he nigericin-containing media.
A detailed protocol can be found at: Steinberg, B.E., and S. Grinstein. 2007. Assessment of phagosome formation and maturation by fluorescence microscopy. Methods Mol. Biol. 412: 289–300.