Isolation and Characterization of sEVs from TNBC Cell Lines

Human metastatic Triple Negative Breast Cancer Cell Lines (TNBC-CL) MDA-MB-231 (CL 1) and MDA-MB-436 (CL 2) as well as a non-malignant cell line, HaCaT (immortalized human keratinocytes), were obtained from ATCC including the authentication certificate (genomic profiling) and were cultured in Dulbecco’s modified Eagle’s medium (Gibco Fisher Scientific). All cell lines were tested for Mycoplasma using Lonza’s MycoAlert detection assay. For TEX production, 4 × 10⁶ cells were cultured in 25 mL medium in a 150 cm² flask for 72 h. The supernatant was collected for sEV isolation as previously described^{37 (link)}. Pre-clarified supernatant was concentrated to 1 mL using a Vivaspin-20, 100,000 MWCO (Sartorius Corp, Bohemia, NY, USA) and placed on a Sepharose 2B column for sEV isolation by size exclusion chromatography (SEC)^{38 (link)}. The Jurkat cell line expressing surface CD8 protein was obtained from Dr H. Rabinowich (Department of Pathology, University of Pittsburgh, PA) and cultured in RPMI medium^{39 (link)}. All culture media were supplemented with 10% (v/v) exosome-depleted and heat-inactivated fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37 °C and in an atmosphere of 5% CO₂ in air.