Grunwald-Giemsa-stained cytospins and by flow cytometry analysis of CD34, CD14, CD66b, CD15, Glycophorin A (GPA), CD41 and CD42b surface antigen expression at day 5, 7, 10, and 12 after the last nucleofection. Images were captured by using an Ax10scopeA1 microscope equipped with AxioCam ERc 5S Digital Camera and Axion software 4.8 (all Carl Zeiss MicroImaging Inc.; Thornwood, NY, USA). The images were then processed with Adobe Photoshop 11.0.2 software.
The following monoclonal antibodies (MoAbs) were used for flow cytometry analysis: phycoerythrin (PE)-conjugated mouse anti-human CD14 MoAb, fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 MoAb, FITC-conjugated mouse anti-human CD66b MoAb, FITC-conjugated mouse anti-human CD15 MoAb (all from Miltenyi Biotech; Auburn, CA, USA), FITC-conjugated mouse anti-human CD41 MoAb, PE-conjugated mouse anti-human CD42b MoAb, and PE-conjugated mouse anti-human GPA MoAb (all from Dako; Milano, Italia). After staining, cells were analyzed by using a BD FACSCanto II (BD Biosciences; San Jose, CA USA). At least 10,000 events were counted for each sample to ensure statistical relevance.
The following monoclonal antibodies (MoAbs) were used for flow cytometry analysis: phycoerythrin (PE)-conjugated mouse anti-human CD14 MoAb, fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 MoAb, FITC-conjugated mouse anti-human CD66b MoAb, FITC-conjugated mouse anti-human CD15 MoAb (all from Miltenyi Biotech; Auburn, CA, USA), FITC-conjugated mouse anti-human CD41 MoAb, PE-conjugated mouse anti-human CD42b MoAb, and PE-conjugated mouse anti-human GPA MoAb (all from Dako; Milano, Italia). After staining, cells were analyzed by using a BD FACSCanto II (BD Biosciences; San Jose, CA USA). At least 10,000 events were counted for each sample to ensure statistical relevance.