May grunwald giemsa staining

Cord blood (CB) was obtained after informed consent from healthy full-term placentas according to institutional guidelines. Human CD34+ cells were purified from CB by positive selection using the midi-MACS immunomagnetic separation system (Miltenyi Biotec, Bergisch Gladabach, Germany) according to the manufacturer’s instructions. The purity of CD34+ cells was assessed by flow cytometry using a monoclonal PE-conjugated anti-CD34 antibody and was routinely over 95 % (range comprised between 92–98 %). Purified human hematopoietic progenitor cells were grown in serum-free medium containing BSA (10 mg/ml), pure human transferrin (1 mg/ml), human low-density lipoproteins (40 μg/ml), insulin (10 μg/ml), sodium pyruvate (10–4 M), l-glutamine (2 × 10⁻³ M), rare inorganic elements (Sn, Ni, Va, Mo, and Mn) supplemented with iron sulfate (4 × 10⁻⁸ M), and nucleosides (10 μg/ml each). HPCs were induced into specific granulopoietic differentiation with IL-3 (1 unit/ml), granulocyte/monocyte CSF (0.1 ng/ml), and saturating amounts of G-CSF (500 units/ml). The differentiation stage of unilineage cultures was evaluated by MayGrunwald–Giemsa staining (Sigma-Aldrich, St. Louis, MO, USA) and cytologic analysis.

Erythroid differentiation was performed by flow cytometry using anti-Ter119 and anti-CD71 antibodies (BioLegend, San Diego, CA) as described previously (Zhang et al., 2018 (link)) on FACS LSR II (BD Biosciences, San Jose, CA). 4′,6-diamidino-2-phenylindole (DAPI, Roche Diagnostics, Basel, Switzerland) was used to exclude the dead cells. Data were analyzed with FlowJo (Tree Star, Ashland, OR). Erythroid differentiation was also analyzed by cell morphology on cytospin slides stained with May-Grunwald/Giemsa staining (Sigma-Aldrich, St. Louis, MO). Cell proliferation was determination by counting nucleated cells daily using crystal violet stain.