Microtubules The microtubule cytoskeleton was visualized using antibody to ��-tubulin. Cells fixed in www.selleckchem.com/products/lapatinib.html PFA (3%, 30 min) and permeabilized in baths with increasing concentrations of alcohol were incubated with mouse monoclonal anti-��-tubulin antibody (1:50). The antigen-antibody complexes were revealed after incubation with anti-mouse IgG coupled with TRITC (1:100). Some reactions were also performed on cells previously treated with nocodazole to check for microtubule depolymerization. MTOCs were detected using mouse monoclonal antibody to ��-tubulin (1:50) according to the method described above for the detection of ��-tubulin. Controls The specificity of the immunocytochemical reactions was determined by incubating cells solely with the anti-rabbit IgG coupled with FITC or anti-mouse IgG coupled with TRITC.
Immunofluorescence samples were mounted in Fluorostab medium (ICN Biomedicals; Aurora, OH) and examined by confocal microscopy with an argon laser (488 nm) for FITC excitation and a helium laser (543 nm) for TRITC excitation. Serial optical sections were collected in the Z axis at 1-��m intervals. Results Growth of CFPAC-1 and CFPAC-PLJ-CFTR6 Cells CFPAC-1 and CFPAC-PLJ-CFTR6 cells displayed differences in their morphology and their growth in culture. In both lines, confluence was reached by day 5 and dome-like structures were observed. CFPAC-1 cells exhibited heterogeneous morphology. Two types of cells were observed: (a) epithelial cells (the majority) with an average diameter of 29 ��m; and (b) fibroblast-like cells grouped in rows.
On average, the latter measured 57 ��m in length and 12 ��m in width. In the CFPAC-PLJ-CFTR6 line, whole cells exhibited epithelial morphology, with a diameter ranging from 10 ��m to 21 ��m. For both cell lines, occludin immunoreactivity was seen as a border around the cells in the apical regions (Figures 1a and and1b),1b), confirming the polarized state. CFPAC-1 and CFPAC-PLJ-CFTR6 cells, plated at a concentration of 1.7 �� 105 cells/ml, both presented a doubling time of 18 hr. However, cell density on day 7 differs between the two cell lines: 3.4 �� 106 cells/ml for CFPAC-1 and 6 �� 106 cells/ml for CFPAC-PLJ-CFTR6 cells. By day 4, the mitotic index was not significantly different between CFPAC-1 (4.8%) and CFPAC-PLJ-CFTR6 (4.6%) cells. The rate of multipolar mitosis, producing up to 4 mitotic spindles, was similar in both CFPAC-1 (0.
24%) and CFPAC-PLJ-CFTR6 (0.29%) cells. Figure 1. Demonstration of the polarized state of CFPAC-1 (a) and CFPAC-PLJ-CFTR6 (b) cells by revealing the tight junction using anti-occludin antibodies. Focal planes passing through the apical regions of GSK-3 cells show the presence of an occludin-immunoreactive … The morphology and growth of the CFPAC-PLJ6 cells were comparable to those observed on the CFPAC-1 cells.