Macs cd34 isolation kit

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 obtain HSC, umbilical cord blood (UCB) samples were obtained with the support of the Department of Gynecology and Obstetrics of Hospital Universitario San Ignacio (Bogota, Colombia) from normal full-term deliveries after obtaining informed consent in accordance with the guidelines approved by the Hospital Ethics Committee. Mononuclear cells were isolated by Ficoll density gradient centrifugation (Histopaque d=1.077 g/cm³, Sigma-Aldrich), and CD34+ cells were isolated using a previously described magnetic-activated cell-sorting (MACS) CD34 isolation kit (Miltenyi Biotec) (15 (link)). Cell purity was evaluated by flow cytometry, and cell viability was determined using trypan blue staining.