Fixable viability dye efluor 450

Freshly isolated TNBC biopsies were digested with collagenase IV (40 mg/mL, Gibco, #17104-019) and filtered through a 100 μm stainless strainer to obtain single-cell suspensions. The cells were resuspended in PBS containing 0.5% BSA and 2 mM EDTA. The prepared single-cell suspensions were blocked with an Fcγ receptor blocker and then incubated with fluorescence-labelled antibodies on ice for 15 min for surface staining. After washing twice with PBS, the cells were fixed and permeabilised with a Cytofix/Cytoperm Kit (BD, #554717) and then incubated with fluorescence-labelled antibodies for intracellular staining. After washing twice with PBS containing 0.5% BSA, the cells were resuspended in PBS containing 0.5% BSA and 2 mM EDTA. A flow cytometer (Thermo Fisher Attune NxT) was used to measure the events. The antibodies used in this study included PE anti-CD31 (Biolegend, #303106), Fixable Viability Dye–eFluor 450 (Biolegend, #65-0863-14), 7-AAD (Invitrogen, #A1310), APC anti-human CD298/ATP1B3 (Biolegend, #341706), FITC anti-HSPA1B (Cusabio, #CSB-PA28047C0Rb), Alexa Fluor anti-KRT7 (Novus, #NBP2-47944 AF700), APC anti-CD93 (Biolegend, #336120), and FITC anti-CDH5 (LSBio, #LS-C467144-100).