Confocal microscopic imaging of single cells.

<p>All images shown are from cultures of 143B cells treated with STS for 24 h. (A) Representative images of cells permeabilized with digitonin, showing retention or redistribution of cyt c or Smac from mitochondria. Cells were processed as for flow cytometry, then immunostained with antibodies specific for cyt c and Smac. Cells were then stained with DAPI (to visualize the nucleus) and finally centrifuged on coverslips prior to confocal microscopy. Images were acquired by confocal microscopy using a FluoView 500 with 60x objective lens. Overlay 1 is a merge between DAPI stain (blue channel) and cyt c (red channel); Overlay 2 is a merge between DAPI stain (blue channel) and Smac (green channel). (B) Representative images of 143B cells subjected to direct immunocytochemical procedures with antibodies specific for cyt c and Smac, and also DAPI-stained, showing retention or redistribution of cyt c or Smac. Cells were grown in 6-well plates, then centrifuged to recover both detached and adherent cells, which were then fixed with 3.5% paraformaldehyde and treated with Triton X-100 prior to immunostaining. Slightly different nomenclature is used to describe the complementary attributes of the top rows of cells in (A) and (B) because flow cytometry detects cells that retain the target protein while direct immunocytochemistry scoring is based on the observed redistribution of target proteins from mitochondria to the cytosol (but still within the boundary plasma membrane). Arrow indicates relevant cell in Row B2. Scale bar (20 µm) applies to all images in each of panels A and B. Very occasionally, significant amounts of Smac may appear in nucleus (cf. ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042298#pone.0042298-Kim1" target="_blank">[29]</a>), but this is extremely rare, with less than 3% of cells showing such localization of Smac (data not shown).