H2 dd pe

To assay maternal cells’ presence in the pups’ lymphoid organs, cell suspensions were prepared from postnatal day 13-15 pups’ spleen and thymus by mechanical dissociation using 70 μm nylon mesh (Axel). Cell suspensions were treated with red blood cells lysis buffer (ammonium-chloride-potassium) and washed in staining buffer [HBSS(-), 2 mM EDTA, 1% BSA]. Single cells were stained in a 96-well plate at a concentration of 3-5×10⁶ cells per well. Cells were stained simultaneously with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (eBioscience) at 1 μl/well, H2Kb-FITC (BioLegend clone AF6-88.5) and H2Dd-PE (BioLegend clone 34-2-12) at a concentration of 1/75th for 30 min at 4°C; fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences); blocked with Fc Receptor Binding Inhibitor Polyclonal Antibody (eBioscience) at 20 μl/well for 15 min and stained intracellularly with HB-EGF-APC (R&D Systems clone 125923) at a concentration of 1/10th for 30 min at room temperature. The following day, 20-30×10⁶ cells per sample were analyzed and sorted on a fluorescence-activated cell sorter (BD FACSAria III) using the BD FACSDiva software (Becton-Dickinson). Sorting was performed by gating for live singlets and maternal cells’ markers on gates H2Kb⁺H2Dd^-Hbegf⁺ and H2Kb^intH2Dd⁺Hbegf⁺ for F1 and F2 pups, respectively, and the ratio of sorted cells over live cells R1 was recorded.