Indirect immunofluorescence analysis of thrombasthenic CD34+ cells transduced with −889PlA2β3.
The RSΔ9β3 and EAY115Cβ3CD34+ cells were transduced with −889PlA2β3, induced to form megakaryocytes ex vivo, and then examined by indirect immunofluorescence analysis for αIIbβ3surface expression. Cells were blocked in 2% BSA, and incubated with 5 μg monoclonal antibody 6D1 that recognizes megakaryocyte-specific glycoprotein (GP)Ib (top panels) or 5 μg AP2, which recognizes αIIbβ3 (bottom panels), and detected with a PE-conjugated F(ab′)2 donkey antimouse secondary antibody. Five to 10 days after transduction, megakaryocytes were present in untransduced and β3-transduced cell cultures as demonstrated with the anti-GPIb antibody; however, only β3-transduced cells demonstrated detectable αIIbβ3 complex on the surface of derived megakaryocytes similar to cultured megakaryocytes from a normal individual (control). There are at least 3 cells in each field of untransduced thrombasthenic cells stained with AP2 for αIIbβ3.
