Pe conjugated anti cd127

Mice tibias were harvested, the epiphyses of the bones were cut and immersed in 15 ml conical tubes with 1.0 ml PBS. Total BM cells were collected by centrifugation at 3,000×rpm for 10 min, and red blood cells were removed with lysis buffer. For HSPC subsets detection, 10⁶ cells were stained with FITC-conjugated anti-Lin, PE-Cy7-conjugated anti-Sca-1, APC-conjugated anti-CD34, Brilliant Violet 421™-conjugated anti-CD16/32, PE-conjugated anti-CD127 (IL-7R) or Brilliant Violet 510™-conjugated anti-CD127, APC-Cy7-conjugated anti-CD117 (c-Kit), PerCP-Cy5.5-conjugated anti-CD90.1 (Thy1.1), PE-Cy5-conjugated anti-CD135 (Flk2) or PE-conjugated anti-CD135 (Biolegend, San Diego, California, USA) for 20 min at room temperature protected from light. Samples were then washed again before resuspension in 0.5 ml PBS containing 2% FBS. Acquisition was conducted on an Attune™ NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). All the data were analyzed as following: Positive cells events (%) = (the events in target gate/the total cell) × 100.

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: fluorescein isothiocyanate (FITC)-conjugated anti-HLA-DR; phycoerythrin (PE)-conjugated anti-CD86; and allophycocyanin (APC)-conjugated anti-CD83 (Miltenyi Biotec). APC-conjugated anti-CD3; and Alexa Fluor 488-conjugated anti-IFN-γ (BD Pharmigen). PE-conjugated anti-IL-10; Alexa Fluor 488-conjugated anti-IL-17A; peridinin-chlorophyll-protein (PerCP)-conjugated anti-CD4; Alexa Fluor 488-conjugated anti-FOXP3; PE-conjugated anti-CD127; and APC-conjugated anti-CD25 (Biolegend). Cells were washed with PBS/EDTA 2 mM/0.5% BSA and stained for 15 min at room temperature in the darkness. For analysis of FOXP3 expression in human T cells primed with hmoDCs, cells were first subjected to surface staining with anti-human CD127-PE, CD4-PerCP, and CD25-APC antibodies. After fixation and permeabilization, cells were stained with anti-human FOXP3-Alexa Fluor 488, according to manufacturer’s recommendations. For each staining, the corresponding isotype controls (IgG2A-FITC, IgG1-Alexa Fluor 488, IgG1-PE, IgG2A-PerCP, or IgG1-APC) were also assayed. Flow cytometry analysis was performed in a FACSCalibur in the Cytometry and Fluorescence Microscopy Unit at Complutense University of Madrid.