Apoptosis-induced membrane PR3 expression on cytoplasts: a model of granule-free neutrophils. (A) Morphology of cytospin, freshly isolated neutrophils and neutrophil-derived cytoplasts. Nuclei are visible in MGG-stained neutrophils but not cytoplasts (magnification ×40). After methanol permeabilization of all membranes, including those of granules, MPO was detected only in neutrophils by immunolabeling and phase-contrast microscopy. PR3 and αII-spectrin immunolabeling of neutrophils and cytoplasts after SLO permeabilization. Slides were analyzed by confocal microscopy with a Zeiss LSM-5 confocal scanning laser microscope version 3.2 SP2 (Zeiss), equipped with an argon laser and helium-neon lasers, using a plan-aprochromat ×63/1.40 NA oil immersion objective lens and a crop ×2, at room temperature. Green fluorescence was observed with a 505-530–nm band-pass emission filter under 488-nm laser illumination and red fluorescence with a 560 long-pass emission filter under 543 laser illumination. Pinhole diameters were set to get 0.8-μm thick optical slices, and images were collected every 0.4 μm along the z-axis. Imaging corresponding to alexa 488, alexa 555, and rhodamin fluorescence were obtained using the multitrack mode. (B) Quantification of PS externalization assessed by annexin-V positivity (and 7-AAD negativity) of neutrophils and cytoplasts by flow cytometry, under basal conditions or after physiologic or gliotoxin-induced apoptosis. Under basal conditions almost no PS externalization was detected on either neutrophils or cytoplasts, whereas after apoptosis induction, significantly more (*P < .05 ANOVA) annexin-V–labeled neutrophils and cytoplasts were observed. Data are mean (± SEM) of the percentage of positive cells in neutrophils from 6 distinct donors. (C) Flow-cytometry analysis of membrane PR3 expression (bold lines) on neutrophils and cytoplasts versus control IgG1 (thin lines). In this representative experiment, under resting conditions (basal), 82% of neutrophils or their corresponding cytoplasts expressed PR3 at their cell surface. Under basal and apoptotic conditions, respectively, the MFIs of membrane PR3 expression were 46 and 190 on neutrophils and 51 and 116 on their corresponding cytoplasts. (D) Flow-cytometry analysis of degranulation markers CD35, CD66b, and CD63 (bold lines) versus isotypic control (thin lines) on cytoplasts under basal or apoptotic conditions. Panels A, C, and D report representative results for neutrophils from a given donor and their corresponding cytoplasts, which were confirmed in independent experiments performed with 3 different cytoplast preparations from 3 different donors.