|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. Quantitative analysis of phagosome-lysosome fusion by flow cytometry. THP-1 cells were differentiated with PMA in the presence of 1 mg/ml F-DXT then washed and chased for 4-5 hours. F-DXT-loaded cells were stained with PKH and allowed to ingest latex beads for the indicated times. (A) Adherent cells on coverslips were then examined by fluorescence microscopy at 2 hours post phagocytosis, as described in Fig. 1A. Color images of merged green (F-DXT) and red (PKH) fluorescence signals show colocalization (yellow), which indicates substantial fusion between phagosomes containing latex beads and F-DXT-loaded lysosomes. The inset shows isolated phagolysosomes in post-nuclear cell homogenates, which were examined by flow cytometry in B. Bar, 5 µm. (B) Phagosomes were prepared 30 minutes, 1 hour and 2 hours after phagocytosis and analyzed by flow cytometry. Green fluorescence was analyzed on positive red fluorescent events, which corresponded to particles surrounded by a phagosomal membrane (gate R3), and the results were expressed as green fluorescence histograms. In each panel, histograms on the left represent PKH-labeled phagosomes isolated from cells without F-DXT loading, and histograms on the right represent phagosomes from cells loaded with F-DXT. The frequencies of phagosomes colocalizing with F-DXT and MFIs increased as a function of time. Values represent the averages of two independent
|
