P55CDC dynamics and localization in endomitotic megakaryocytes.

Immunofluorescence experiments were performed on human megakaryocytes at day 5, following CD34⁺ cells cultured with PEG-rhuMGDF and SCF. Cells were cytocentrifuged, fixed with paraformaldehyde, and permeabilized with triton. (A) p55CDC immunostaining was performed by using a rabbit antibody followed by incubation with donkey FITC-labeled antirabbit (in green), and DNA was counterstained with TOTO3-iodide (in blue). (B) Triple immunostaining was performed by using rabbit anti-p55CDC, mouse antitubulin, and human antikinetochore antibodies, followed by incubation with donkey FITC-labeled antirabbit (in green), TRITC-conjugated antimouse (in red), and Cy-5 labeled antihuman (in blue), F(ab′)2 fragments, respectively. (C) Double immunostaining anti-p55CDC (in green) and antitubulin (in red) and nuclei counterstained with TOTO3-iodide (in blue). In A, B, and C, cells were examined by confocal microscopy with a 3-dimensional reconstruction and superimposition of the markers. Triple staining merge is shown on the first column. (A) Endomitotic megakaryocyte at prophase. (B) 8N to 16N endomitosis at prometaphase, metaphase, anaphase. P55CDC and kinetochore signals colocalization provides a fusion pale-blue signal (corresponding magnification). (C) Megakaryocyte of high ploidy at the end of an endomitosis. Bar, 5μm.