Abstract
The pH of endosomes can be measured with minimal interference to cellular function using internalized ligands or antibodies labeled with pH sensitive fluorescent probes. The most convenient indicator for such measurements is Fluorescein Isothiocyanate (FITC), and numerous specific antibodies and ligands labeled with FITC are readily available. Covalent labeling of specific proteins or antibodies with FITC is relatively easy to perform at low cost. Measuring FITC fluorescence ratio involves placing a drop of oil on the immersion objective and position the loaded Leiden dish in an open perfusion microincubator over the objective. Turn on the computer and other external hardware devices. Verify that the temperature controller of the microincubator is set to the appropriate value. At the end of the acquisition period, select one representative field and perfuse the dish sequentially with the calibration solutions. Monitor the fluorescence to ensure establishment of a stable ratio, indicating equilibration of the vesicular pH with the external pH.
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