Fig. 4.
Fig. 4. Adhesion to FN. / Adhesion to FN leads to increased levels of p27KIP1 and decreased levels of cyclin-E. Mobilized blood CD34+ cells, cultured for 48 hours in serum-free medium with low-dose cytokines, were incubated with the activating anti–β1-integrin antibody 8A2 for 30 minutes at 37°C and plated in PLL- or FN-coated dishes for 12 to 16 hours. Adherent and nonadherent cells were collected separately. Cells were fixed, permeabilized, and incubated with antibodies directed at p27KIP1 and cyclin-E (open histogram) or isotype control (closed histogram) antibody at room temperature for 30 minutes followed by FITC-conjugated goat antimouse immunoglobulin for 30 minutes in the dark. Cells were washed and resuspended in 50μg/mL propidium iodide. Cells were analyzed on a FACS-Calibur flow cytometer with the use of CellQuest software. A representative example of 5 individual experiments is shown.

Adhesion to FN.

Adhesion to FN leads to increased levels of p27KIP1 and decreased levels of cyclin-E. Mobilized blood CD34+ cells, cultured for 48 hours in serum-free medium with low-dose cytokines, were incubated with the activating anti–β1-integrin antibody 8A2 for 30 minutes at 37°C and plated in PLL- or FN-coated dishes for 12 to 16 hours. Adherent and nonadherent cells were collected separately. Cells were fixed, permeabilized, and incubated with antibodies directed at p27KIP1 and cyclin-E (open histogram) or isotype control (closed histogram) antibody at room temperature for 30 minutes followed by FITC-conjugated goat antimouse immunoglobulin for 30 minutes in the dark. Cells were washed and resuspended in 50μg/mL propidium iodide. Cells were analyzed on a FACS-Calibur flow cytometer with the use of CellQuest software. A representative example of 5 individual experiments is shown.

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