Anti cd10 pe cf594

The enumeration of HSPC was conducted in whole UCB using BD Trucount tubes (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instruction. 100 µl whole UCB were added by reverse pipetting to BD Trucount tubes and stained with anti- CD45− FITC, anti- CD34− PE, anti- CD38− PE- Cy7, anti- CD10− PE- CF594, 7-AAD (all BD Biosciences) and anti- CD133– APC (Miltenyi Biotechnology, Bergisch, Gladbach, Germany). After immunofluorescence staining, erythrocytes were lysed with ammonium chloride lysis solution and analyzed within one hour by flow cytometry (LSRII, BD Biosciences) and analysed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For enumeration of HSPC in UCB, CD34+ cells were gated according to the modified ISHAGE criteria (CD45^dim/7-AAD-/CD34+ cells). CD10 was added to exclude B- lymphoid progenitors. The number of cells/µl was calculated as [(# gated cells/# acquired beads) * (# of beads per test/test volume).

For flow-cytometric analysis hematopoietic cells and ECs were stained with different combinations of monoclonal fluorochrome-conjugated antibodies (see Supplemental Table 5) for at least 20 min at 4 °C. Propidium-Iodide (PI) or 7-Aminoactinomycin D (7-AAD) were used for dead cell exclusion. Appropriate isotype-matched, control monoclonal antibodies were used to determine the level of background staining in all experiments. Flow cytometric analyses were performed on a FC500 flow cytometer equipped with the CXP 2.2 software (Beckman Coulter) (Fig. 1C). Cells were sorted using a FACSAria I cell sorter. The sort-purity was routinely assessed by recovery of sorted cells and was >99.5%.
Samples from murine BM were stained with the following fluorochrome conjugated antibodies: anti-CD34-APC-AF750 (Beckman Coulter), anti-CD133-APC (Miltenyi Biotec), anti-CD45RA-BV711 (BioLegend), anti-CD38-BV785 (BD Biosciences), anti-CD10-PeCF594 (BD Biosciences) and CD45-BV510 (BD Biosciences). DAPI staining was used for excluding dead cells. All measurements were performed in a BD FACSAria^TMIII (Beckman Coulter) flow cytometer.