Stat1−/− phagosomes show a selective defect in phagosomal acidification.

<p>(A) Phagosomal pH-measurement. Monolayers of untreated (left panel) or LPS/IFNγ treated (right panel) SV129 control and Stat1−/− PEMs were established on 1 by 2.5 cm glass slides and pH of phagosomes containing FITC-labeled zymosan was determined by ratio-fluorescence determination. Calibration for fluorescence was done in situ. The fraction of particles bound to and remaining on the cell surface was controlled for and found to be negligible by measurement of the response to alterations in the pH of the external buffer. Each experiment was performed in duplicate and repeated at least three times. The graph shows a representative time course measurement of phagosome alkalinization upon addition of 10 µM NH₄Cl (open arrow head), equilibration and removal (closed arrow head). (B) and (C) Measurement of intracellular pH. (B) Monolayers of SV129 control and Stat1−/− PEMs were incubated for 30 min with 10 µM BCECF-AM, overnight with 2.5 mg/ml FITC-dextran, or 2 h with FITC-labeled zymosan, and cytoplasmic, lysosomal, or phagosomal pH were assessed by ratio-fluorescence determination. The bars represent the standard deviation of three independent duplicate experiments. (C) Monolayers of SV129 control and Stat1−/− PEMs were infected for 2 h with FITC-labeled axenic <i>L. donovani</i> amastigotes (Leish) or incubated with FITC-labled zymosan (zymo), and phagosomal pH were assessed by ratio-fluorescence determination.