Facs influx instrument

The fourth pair of lactating mammary glands were harvested from the mice and the inguinal lymph nodes carefully removed from the tissue preparation. Each mammary gland was finely sliced and digested with Liberase at 83 µg/mL and DNAse I at 40 µg/mL in HBSS, 0.5% BSA, 10 mM Hepes for 20 min at 37°C. Digested preparations were passed through filters of diminishing grid size (100, 70, and 40 µm), crushed between two filters, and washed three times. The number of cells of each lactating mammary gland preparation were determined, adjusted to 10 x 10⁶ cells per mL, and incubated with FcR Blocking Reagent (Miltenyi Biotec) for 10 min on ice to avoid non-specific labelling through Fc-receptors. Lactating mammary gland cell suspensions of each mouse were labeled using TotalSeq™ anti-mouse hashtag 1-3 antibodies A301-303 (Biolegend, UK) for 30 min at 4°C. After two washing steps, the three suspensions were pooled and stained with anti-IgG2b-Vioblue antibody to specifically label CD45 cells. Cell suspensions were then labeled with 7AAD (Biolegend) as a viability marker. Doublet, 7AAD^pos and FITC^pos cells were gated out to sort only viable resident CD45^pos immune cells using a BD FACS Influx instrument (BD Biosciences) and 90,000 cells were recovered.

Cells from PDOs were prepared using 300 units/mL collagenase IV (Worthington, LS004210) at 37°C for 30 minutes, followed by three 5-minute washes with RPMI-1640 (Corning) supplemented with 10% FBS (MilliporeSigma) and 50 units/mL penicillin/streptomycin (Beyotime). Cells were treated with BD Fix Buffer I and Perm Buffer III and were stained for 45 minutes at room temperature with the following antibodies: PE-conjugated anti-CD133 antibody (eBiosciences, 12-1338-42), anti–human OPA1 antibody (Cell Signaling Technology, 67589), and anti–human SPDEF antibody (Santa Cruz Biotechnology Inc., sc-166846). BODIPY 493/503 (Thermo Fisher Scientific, D3922) was used to quantify the intracellular accumulation of lipid droplets. Cells were analyzed using BD Canto II FACS analysis, and for some experiments, live cells were sorted with the BD FACS Influx instrument. Fluorescence minus one (FMO) was used for the gating strategy. Data were analyzed with FlowJo software.