CRU assays were performed using limiting numbers of CD34+ cells from human cord blood (Lonza). Freshly isolated non-cultured human cord blood CD34+ cells were immediately used for BM transplantation, or human cord blood CD34+ cells were co-cultured with murine rMSCs for 6 days in StemSpan SFEM media (STEMCELL Technologies) supplemented with 10 % KnockOut Serum Replacement (Thermo Fisher Scientific), Pen/Strep, 10ng/ml of human TPO (PROSPEC) and 20ng/ml of human SCF (PROSPEC), then collected and human CD45+ cells were isolated by MACS column (Miltenyi Biotec), and finally a fraction of the cultured cells corresponding to the indicated number (0.2, 1, 3 and 5 × 103) of initial cord blood cells was subjected to BM transplantation. The test cells (human CD45+) were transplanted into NOD-scid Il2rg−/− (NSG) immunocompromised mice sublethally irradiated (2 Gy) together with 2 x 105 competitor BMNCs (NSG). The percentage of donor (human CD45+) cells in the PB was determined 16 weeks later by flow cytometry. Mice with more than 1% were considered positive and the others were defined as negative mice. The CRU frequency was calculated using L-Calc software (STEMCELL Technologies).
Human cord blood
Manufactured by Lonza
Human cord blood is a biological sample collected from the umbilical cord and placenta shortly after birth. It contains a variety of cells, including hematopoietic stem cells, which can be used for research and therapeutic applications.
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2 protocols using human cord blood
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Cord Blood CD34+ Cell Transplantation Assay
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Cord Blood CD34+ Cell Transplantation Assay
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CRU assays were performed using limiting numbers of CD34+ cells from human cord blood (Lonza). Freshly isolated non-cultured human cord blood CD34+ cells were immediately used for BM transplantation, or human cord blood CD34+ cells were co-cultured with murine rMSCs for 6 days in StemSpan SFEM media (STEMCELL Technologies) supplemented with 10 % KnockOut Serum Replacement (Thermo Fisher Scientific), Pen/Strep, 10ng/ml of human TPO (PROSPEC) and 20ng/ml of human SCF (PROSPEC), then collected and human CD45+ cells were isolated by MACS column (Miltenyi Biotec), and finally a fraction of the cultured cells corresponding to the indicated number (0.2, 1, 3 and 5 × 103) of initial cord blood cells was subjected to BM transplantation. The test cells (human CD45+) were transplanted into NOD-scid Il2rg−/− (NSG) immunocompromised mice sublethally irradiated (2 Gy) together with 2 x 105 competitor BMNCs (NSG). The percentage of donor (human CD45+) cells in the PB was determined 16 weeks later by flow cytometry. Mice with more than 1% were considered positive and the others were defined as negative mice. The CRU frequency was calculated using L-Calc software (STEMCELL Technologies).
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