Alexa fluor 488 conjugated antibody a11029

The cells, seeded on coverslips, were fixed in 4% buffered paraformaldehyde solution, permeabilised using 0.1% TritonX-100, incubated overnight with rabbit anti-pH3 antibody 04-817 (Millipore), washed with PBS containing 5 mM EGTA and incubated with goat anti-rabbit Alexa Fluor 594-conjugated antibody A11012 (Invitrogen) for 1 h at RT. For subsequent staining of tubulin, cells were washed with PBS-EGTA, incubated with mouse anti-tubulin antibody T5168 (Sigma) for 1 h at RT, washed with PBS-EGTA and incubated with goat anti-mouse Alexa Fluor 488-conjugated antibody A11029 (Invitrogen). For counterstaining of cell nuclei, the coverslips were mounted using Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Cells were examined using an Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany).

Cell viability was determined with the alamarBlue® assay (Invitrogen) as described in [47 ]. Cell cycle phase distribution was analyzed by flow cytometry using propidium iodide (PI) as described in [14 (link)]. For M-phase quantification, cells were fixed with 70% ethanol, permeabilized using 0.25% TritonX-100, incubated with anti-phospho-histone H3 (pH3) antibody 06-570 (Millipore, Temecula, CA) for 1 h at room temperature (RT), washed with PBS containing 1% bovine serum albumin (BSA) and incubated with goat anti-mouse Alexa Fluor 488-conjugated antibody A11029 (Invitrogen) and PI. Following analysis with flow cytometry, the data were processed with the Cell Quest software (Becton Dickinson, San Jose, CA) or ModFit Software (Verity Software, Topsham, ME). For the analysis of cell cycle phase distribution of cells from tumor and lung tissue of A549 xenografts, the formalin-protease method was adopted as described [48 (link)], followed by quantification using ModFit Software. Apoptosis was studied using Annexin V/PI method (Annexin V-FLUOS staining kit, Roche, Mannheim, Germany) according to the manufacturer's protocol. Flow cytometric analysis was immediately performed using FACS Calibur.