Pe conjugated cd34 antibody

CD34⁺ progenitors were isolated by immunomagnetic bead selection on an
affinity column using magneticactivated cell-sorting (MACS) CD34 Isolation Kit (130046702,
Miltenyi Biotec, Bergisch-Gladbach, Germany). Briefly, the MNCs were suspended in PBS (up
to 108 total cells). Then, 100 µl human Fc receptor (FcR) blocking reagent and 100 µl
anti-CD34 microbeads were added, mixed and incubated at 4°C for 30 minutes. Subsequently,
the MNCs were washed with PBS and centrifuged at 300 g for 10 minutes. The labeled MNCs
were then re-suspended in 3 mL PBS and passed through an LS separation column (130042401,
Miltenyi Biotec, Bergisch-Gladbach, Germany) placed in a magnetic field to capture the
CD34⁺ cells. In order to collect the CD34⁺ cells, the column was
removed from the magnetic field and flushed with a PBS and 2 mM EDTA washing solution. The
purity of CD34⁺ cells was assessed by flow cytometry using antihuman
phycoerythrin (PE) -conjugated CD34 antibody (550761, Pharmingen, San Diego, CA, USA).

To evaluate the engraftment and multi-lineage differentiation of the human CD34+ cells transduced with the hAS8 lentiviral vector, cells from the peripheral blood, bone marrow, thymus and spleen were analyzed from the transplanted NRG mice. Total cells were obtained from the above-mentioned organs and were labeled with antibodies specific for human immune cells including T cells, B cells, macrophages and CD34+ cells. The following antibodies were used in these studies: T cell antibodies used were a Brilliant violet (BV) 421-conjugated CD3 antibody, an allophycocyanin (APC) H7-conjugated CD4 antibody and an Alexa fluor (AF) 700-conjugated CD8 antibody (BD Biosciences, San Jose, CA). B cell antibodies used were a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD19 antibody (BD Biosciences, San Jose, CA). Macrophage antibodies used were a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD14 antibody (BD Biosciences). CD34+ cell antibodies used were a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD34 antibody (BD Biosciences, San Jose, CA). Flow cytometry was performed using a BD Fortessa and analyzed with CXP software.