TaqMan™ Array Human Inflammation 96-well Plate for Caco-2 cell line

Peripheral blood was acquired from the Pécs Regional Blood Transfusion Center (Pécs, Hungary) and processed using Lymphocyte Separation Medium 1077 (Promocell, Heidelberg, Germany) in accordance with the manufacturer's instructions. Subsequently, the viable mononuclear cells were isolated, counted, and seeded into 6-well plates at a density of 1,000,000 cells per well. The culture medium was specifically formulated using RPMI-1640 medium (Biowest, Nuaillé, France), enriched with 1% L-glutamine (Lonza, Basel, Switzerland), 2% penicillin/streptomycin (Lonza, Basel, Switzerland), and 10% exosome-depleted FBS (Thermo Fisher Scientific, Waltham, USA) to minimize EV interference from bovine serum, further supplemented with 100 ng/mL Peprotech M-CSF 300-25 (Gibco, Waltham, USA). The cells were cultured for 5 days in a 5% CO2 incubator at 37 °C, resulting in the differentiation of all monocytes into macrophages (M0) (Cao et al., 2019). Differentiated macrophages were treated with MEV (Milk-derived extracellular vesicles) from healthy and mastitis groups separately to achieve the final concentration of 2×10^11 particles/mL. Additionally, untreated cell cultures with the same volume of PBS were maintained as negative controls. After 48 hours, macrophage supernatants were collected and stored at -80 °C (for a maximum of 3 months) for future experiments. Human colorectal adenocarcinoma Caco-2 cells were purchased from the American Type Culture Collection and were used from passages 52 to 58 following the supplier’s recommendations. Caco-2 cells were cultured in DMEM medium (Biosera, Cholet, France) with 10% exosome-depleted FBS (Thermo Fisher Scientific, Waltham, USA). Cells (30,000 per well) were seeded in 24 well plates one day before treatment. The supernatant from macrophages accounted for 1/3 of the total volume of the Caco-2 culture medium. The experimental groups were divided into negative control, healthy group and mastitis group.