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Fig. 1. Flow cytometry analysis of latex bead phagosome. (A) PKH-labeled THP-1 cells adherent to coverslips and ingesting latex beads (4 µm) were fixed and mounted on microscopy slides. Samples were examined at the optimal wavelengths for PKH26 (551 excitation, 567 nm emission) using a PL-Apochromat 63x/1.4 oil immersion objective on a Zeiss Axioplan II microscope. Latex beads are surrounded by a red fluorescent membrane. Bar, 5 µm. (B) In (a), equal volumes of 1, 2 and 4 µm latex beads were mixed and analyzed by flow cytometry. Beads of different sizes were localized by plotting forward (FS) versus side (SS) scatter channels on a linear scale. (b) Postnuclear supernatant from THP-1 homogenate mixed with 4 µm beads and analyzed by flow cytometry using similar SS and FS parameter settings as in (a). Large latex beads (R1 window) were readily discriminated from cell debris. (d) Red fluorescence (FL2) of partially purified phagosomes from PKH labeled cells that have ingested 4 µm latex beads. Events were recorded from the R1 window, which is the predicted position of phagosomes based on the localization of free beads in (b). (c) Fluorescence of latex beads alone (background autofluorescence), allowing for positioning of window R2 in (d) to include signals from true phagosomes only. A comparison of (c) and (d) shows that a significant fraction of latex beads were released from phagosomes during homogenization and centrifugation. (C) Phagosomes were sorted using a Becton Dickinson FACStar plus flow cytometer on the basis of positive red fluorescence (gate R2 shown in Bd); 
5 million sorted early (30 minutes) and late (2 hours) phagosomes were pelleted and phagosome membrane proteins were solubilized in 1xLaemmli buffer. Samples were separated by SDS-PAGE along with 100 µg of total cell lysate (WCL). Proteins were transferred to nitrocellulose membranes and probed with antibodies against pleckstrin, Rab5 and Rab7.
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