Anti cd45 fitc

Cell cycle analysis was carried out using propidium iodine (PI) staining. In short, after exposure to ⁹⁰SrCl₂, cells were detached by trypsin incubation, washed twice and counted. 300,000 cells were fixed in 70% ethanol and stored at −20 °C for at least one hour. Cells were then centrifuged 8 minutes at 400 g and the pellet was re-suspended with a sodium citrate solution containing 0.2% Triton X-100, 100 μg.mL⁻¹ RNAse and 50 μg.mL⁻¹ PI (Sigma) and incubated 30 minutes at 37 °C in the dark. Stained cells were then analyzed on a FACS Canto II (BDIS, Le Pont-de-Claix, France) with the acquisition of at least 10,000 events per condition.
Phenotypic analysis of rat BMSCs was performed using the following directly coupled antibodies: anti-IgG1-FITC, anti-IgG2a-PE, anti-CD44-FITC, anti-CD45-FITC, anti-CD90-PE, anti-CD73-alexa647 (all from BD Pharmingen, Le Pont-de-Claix, France) and anti-CD34-PE (Santa Cruz Biotechnologies). At the first passage, aliquots of 200,000 cells were washed twice in PBS and incubated for 20 minutes in the presence of the indicated antibodies at pre-defined concentrations. Cells were then washed twice and analyzed.

PHH were thawed, washed once with PBS and immediately fixed with 4% paraformaldehyde for 30 minutes on ice. After fixation, the cells were pelleted and blocked for 30 minutes on ice in PBS supplemented with 5% normal goat serum, 5% normal rabbit serum and 0.1% BSA for staining of cell-surface markers, or according to the manufacturer’s instructions for the eBioscience Intracellular Staining Kit (eBioscience, San Diego, CA) when detecting intracellular antigens. Next, cells were incubated with anti-CD45-FITC, anti-CD81-APC or anti-CD68-FITC (all BD Biosciences, San Jose, CA), anti-HLA-DR-FITC or anti-CD299-APC (both eBioscience), anti-human albumin-FITC (Rockland, Limerick, PA) or isotype-matched controls for 60 minutes on ice. All antibodies were used at manufacturer recommended concentrations except for anti-human albumin-FITC, which was used at 10 μg/mL. After staining, PHH were washed three times with PBS before being assessed by flow cytometry using a Fortessa LSR instrument (BD Biosciences). All flow cytometry data was analyzed using FlowJo V10.08r1 software (TreeStar Inc., Ashland, OR). A detailed description of all antibodies used is provided in S6 Table.

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).