Quantification of Phagosome or Efferosome pH by Ratiometric FITC Imaging

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.

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