Flow cytometric analysis following −889PlA2β3 transduction of thrombasthenic CD34+ cells.
Untransduced and transduced cells were induced to form megakaryocytes for 9 days ex vivo, and then examined by flow cytometric analysis for surface expression of the β3 subunit. Shown in the first set of panels are megakaryocytes expressing αIIbβ3 as detected with complex-specific antibody AP2 and a PE-conjugated F(ab′)2 donkey antimouse secondary antibody. The second set of panels are cells expressing the β3 subunit as detected with monoclonal antibody, AP3, and secondary antibody. Fluorescence contour plots are presented for normal nonthrombasthenic cells (upper right), untransduced and transduced cells from EAY115Cβ3 (middle panels), and untransduced and transduced cells from RSΔ9β3 (lower panels). The x-axis depicts cell size as measured with forward scatter on a linear scale, and the y-axis is relative fluorescence intensity of PE-AP2 or PE-AP3. Megakaryocytes that expressed αIIbβ3 on the cell surface were detected in the upper right quadrant as were cells that expressed β3. Normal and patient cells incubated with an isotype nonspecific antibody and secondary antibody were presented as controls for background staining (upper left panels).
