Journal: Cell Adhesion & Migration
Article Title: Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells
Figure Lengend Snippet: rADAM9D binds to DU145 through β1, α6, αvβ5 and αvβ3 integrins as demonstrated by an antibody competition assay ( A ) and by flow cytometry analysis ( B ). The integrin content of DU145 cell line was assessed by flow cytometry ( C ). ( A ) For antibody competition assay CMFDA-labeled cells were incubated with different anti-integrin antibodies (β1, α6, αvβ5, αvβ3, α2 and α4, at 10 µg/ml) and IgG control (10 µg/ml) before being plated on rADAM9D-coated (10 µg) wells. DU145 directly plated on rADAM9D or IgG-coated wells, without previous incubation with any antibody was used as positive control and BSA was used as negative control. ( B ) The integrin content of DU145 cells was determined by flow cytometry. Cells (1 × 10 5 ) were incubated for 40 min at 4°C with the specific antibodies cited earlier or control IgG. Cells were washed and incubated with secondary antibody labeled with FITC, at same conditions described before, washed and fixed with FACs buffer contaning 1% phormaldehyde overnight at 4°C. C. To verify the interaction with integrins, rADAM9D (1 µM) was previously incubated (30 min at room temperature) with DU145 cells, before the addition of antibodies. Cells were analyzed in FACSCanto. The results were obtained from 3 independent experiments in triplicate. The error bars show the SE of three samples with less deviation from the mean. The means that are significantly different from rADAM9D and IgG-coated wells using ANOVA followed of post hoc Dunnett's test were shown by *( P ≤ 0.001).
Article Snippet: Secondary antibody goat anti-mouse IgG-FITC (Fluorescein isothiocyanate, sc-2010), used for flow cytometry, were purchased from Santa Cruz Biotechnology.
Techniques: Competitive Binding Assay, Flow Cytometry, Cytometry, Labeling, Incubation, Positive Control, Negative Control, FACS