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Fig. 2. Flow cytometry analysis allows the quantitative evaluation of phagosome maturation. PKH-stained cells were allowed to ingest latex particles, and phagosomes were prepared after 30 minutes, 1 hour and 2 hours. Phagosome preparations were fixed/permeabilized and stained with irrelevant antibodies or specific antibodies to LAMP-1 (A) or Rab7 (B). Preparations were washed and stained with FITC-labeled secondary antibodies. After adequate green/red compensations, green fluorescence was analyzed on positive red fluorescent events, which correspond to particles surrounded by a red phagosomal membrane (gate R3), and the results were expressed as green fluorescence histograms. In each panel, the histogram on the left represents phagosomes stained with irrelevant antibodies, and the histogram displaced to the right represents phagosomes stained with specific antibodies. The proportion of phagosomes expressing LAMP-1 (A) and Rab7 (B) increases as a function of maturation. Values represent the averages of two independent experiments.
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