Muc1 pe cy7

Flow cytometry analysis was performed as previously described (53 (link)). Briefly, 10 mL of fresh healthy human BM containing ~5 × 10⁶ cells collected within 3 months of lactation, was centrifuged at 2,000 × g at room temperature for 10 min, washed with PBS, and centrifuged two more times. The cells were first incubated with Live/Dead Fixable Aqua (ThermoFisher) at room temperature for 30 min and subsequently washed twice with PBS. Besides compensation and FMO controls, two sets of cells were immune-stained at room temperature for 30 min in 0.5% BSA-PBS containing a combination of conjugated antibodies (CD45-PerCP-Cy5.5 BD, CD14-V450 BD, MUC1-PE-Cy7 BioLegend, TLR2-APC R&D Systems, TLR10-PE BioLegend) and (CD45-PerCP-Cy5.5, CD14-V450, MUC1-PE-Cy7, TLR1-APC R&D Systems, TLR10-PE), respectively. The cells were washed twice with PBS and fixed with 1% PFA-PBS. The samples were run on a BD LSRFortessa and analyzed using FlowJo software.

The autologous breast cancer cells were stained with HER-2 PE, epithelial cell adhesion molecule (Ep-CAM) PE-Dazzle 594, mucin-1 (MUC-1) PE-Cy7 and integrin alpha 6 (CD49f) APC (Biolegend, USA) as recommended by the manufacturer. The cells were acquired on the LSRII flow cytometer and the data were analysed as indicated previously.