Pe cyanine7 cd38

Frozen UCB units were provided by the Japanese Red Cross Kanto-Koshinetsu Cord Blood Bank (Tokyo, Japan). Isolation of human CD34⁺ cells from frozen UCB samples starts with introduction of thawed UCB units into CliniMACS Prodigy equipment (Miltenyi Biotec). In a closed tube-bag system, UCB cells were washed in Dulbecco's Modified Eagle's Medium (Sigma Aldrich) containing DNase I (Roche), followed by centrifugation (420 g). This medium was replaced by CliniMACS Buffer and MNCs were isolated from the entire unit in a gradient formed using Ficoll-Paque Premium buffer (GE Healthcare; P = 1.084). MNCs were then washed three times in CliniMACS Buffer and mesh filtered. All these steps were conducted in an automated manner directed by an optimized program (available upon request). Beyond this point, manual positive selection was conducted (CD34 MicroBeads kit; Miltenyi Biotec), generally twice, to increase CD34 purity (possibly ∼100%), as analyzed by staining cells with an FITC-conjugated anti–human lineage antibody cocktail, APC-CD34, Pacific Blue-CD45 (BioLegend), and PE/Cyanine7-CD38 (eBioscience).

Cells from PB samples collected by retroorbital bleeding or BM samples were treated with ACK lysis buffer and stained with these dye-conjugated mAbs: Pacific Orange-Ly-6G/6C (Gr-1; Life Technologies), PE-Cyanin5-CD45R/B220, Alexa Fluor 700-CD4, Alexa Fluor 700-CD8a, PE-Cyanin7-CD45.1, APC-cyanin7-CD45.2, Pacific Blue-H2Kd, FITC-H2Kk, FITC-H2Kb, PE-H2Kb, Alexa Fluor 647-H2Kq, Brilliant Violet 510 Streptavidin (BioLegend), Alexa Fluor 700 Sca-1, and APC c-Kit (eBioscience). Data were acquired by FACSAria and analyzed using FlowJo software (Tree Star). Complete blood counts were obtained with MEK-6450 Celltac-α (Nihon Kohden). For flow cytometry analysis of human cell engraftment in NOG mice, BM samples were stained with these dye-conjugated mAbs: PE-HLA-A2, Pacific Blue-human CD45, FITC-conjugated anti–human lineage antibody cocktail, APC-CD34, APC-CD4, APC-CD8, PE-Cyanin7-mouse CD45 (BioLegend), PE-CD33, FITC-CD19 (BD), and PE/Cyanine7-CD38 (eBioscience).