Extrusion of an apoptotic cell from an HBE monolayer as shown by confocal Z-series. This set of images shows late extrusion events (early and middle events are CIL# 37908 and 37909). The bioactive lipid sphingosine-1-phosphate (S1P) is produced by dying cells and activates actomyosin contraction in surrounding cells. The apoptotic, fragmenting nucleus is in blue, the actin-myosin ring at the basolateral surface is in red and S1P is in green.
To induce apoptosis, cultured monolayers were exposed to 1,200 µJ/cm2 UV254 irradiation in a UV series II (Spectroline) and incubated for 2 h before fixation. Cells were fixed with 4% formaldehyde in PBS, permeabilized with 0.5% Triton, blocked and incubated with primary antibody for 1 h (50 µg/ml anti-S1P mAb (LPath Inc.)). Secondary antibody was Alexa Fluor 488 goat anti–mouse IgG. Actin was detected with Alexa Fluor 568–phalloidin (Invitrogen). DNA was detected with 5 µM DRAQ5 (Axxora). Confocal micrographs were obtained using a microscope (TCS SP5; Leica) with a 63x oil lens with an electron-multiplied cooled CCD camera 1,000 x 1,000, 8 x 8 mm2 driven by the IQ software (Andor Technologies). ImageJ was used to stack confocal sections into Z series that were then color combined and reconstructed into 3D image using MetaMorph (GE Healthcare). All images were processed further using ImageJ, Photoshop (Adobe), Illustrator (Adobe), and Quicktime Pro (Quicktime) software. This Z-series corresponds to Fig 4C from J Cell Biol. 2011. 193(4): 667-676
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