Rmpi 1640 culture medium

For EV isolation, the conditioned medium was filtered at 0.22 μm to remove cellular debris, and then EVs were isolated from the supernatant by ultracentrifugation at 100,000× g for 16 h at 4 °C using Sorvall WX Floor Ultra Centrifuge with AH-629 36 mL swinging Bucket Rotor (Thermo Fisher Scientific, Waltham, MA, USA). Isolated EVs were resuspended with PBS at concentrations of 5 to 10 × 10¹⁰/mL. The particle size and number of EVs were analyzed using the NanoSight LM 10 Nanoparticle Tracking Analysis System (Malvern, Malvern, UK). For in vivo biodistribution assays, iEVs were labeled with a near-infrared fluorescent dye, DiR (ThermoFisher), as reported in [17 (link),43 (link)]. To determine types of iEV recipient cells, iEVs were labeled with a fluorescent dye, PKH26 (Sigma), as reported in [44 (link)]. Splenocytes were isolated from NOD.B10.H2^b mice, cultured with RMPI 1640 culture medium (Gibco, Billings, MT, USA) containing 5% FBS, treated with 3 × 10⁹ particles/mL PKH26-labeled iEVs, and then examined with flow cytometry for PKH26 signal and markers of macrophages, T cells, or B cells as detailed below.

Human esophageal epithelial cells (HEEC) and human EC cell lines (KYSE-520 and ECA-109) were purchased from BioVector NTCC Inc. (Beijing, China). These cells were cultured with RMPI-1640 culture medium (Gibco BRL, Paisley, UK) containing 10% fetal bovine serum (FBS) in a humidified incubator (37°C, 5% CO₂) [32 (link)]. Cells were passage when the confluence until to 80%.

The human melanoma cell lines A375 and M14 were cultured in DMEM culture medium (Gibco BRL, Gaithersburg, MD, USA), containing 10% fetal bovine serum (FBS), where the murine cell line B16F10 and the human melanoma cell line SK-Mel-2 were cultured in RMPI1640 culture medium (Gibco BRL), containing 10% FBS in a humidified incubator at 37 °C and 5% CO₂. α-Mangostin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO in the concentration of 10 mmol/L and stored at 4°C, whereas Cisplatin (Sigma-Aldrich) and dacarbazine (Sigma-Aldrich) were dissolved in phosphate buffered saline (PBS) at 10 mmol/L and stored at -20°C until use. Paclitaxel (Selleck Chemicals, Houston, TX, USA) and vincristine (Selleck Chemicals, USA) were dissolved in DMSO at 10mmol/L and stored at -80°C.

Human umbilical vein endothelial cells (HUVECs, PCS‐100‐013, ATCC USA) were cultured in EC growth media with SupplementMix (PromoCell, Germany) at 37°C, 5% of CO₂ and cells of passage 4‐8 were used. The human monocytes THP‐1 (TIB‐202, ATCC USA) were cultured in RMPI 1640 culture medium (Gibco, USA) supplemented with 10% of FBS and the cell density was kept between 1 × 10⁶ and 5 × 10⁶ cells in a 25 cm² flask.

Neutrophils were obtained from heparinized peripheral blood of healthy human donors (Etablissement Français du Sang, Strasbourg, France). Neutrophils were isolated by negative selection using the MACSxpress kit for the isolation of human neutrophils from whole blood (Miltenyi Biotec, Cat.No. 130-104-434, Paris, France) and the magnetic separator (Miltenyi Biotec, Cat.No. 130-098-308, Paris, France). The supernatants containing neutrophils were collected and the removal of contaminating erythrocytes was performed by positive selection using the MACSxpress human erythrocyte depletion kit (Miltenyi Biotec, Cat.No. 130-098-196, Paris, France) using the manufacturer’s instructions. Neutrophils were suspended in RMPI 1640 culture medium (Gibco, Cat.No. 11875085, Pasley, Scotland). Neutrophil viability was assessed using acridine orange/propidium iodide labelling (Logos Biosystem, Cat.No. F23001, Villeneuve-d’Ascq, France), and their counts measured using a LUNA-FL™ dual fluorescence cell counter (Logos Biosystem, Cat.No. L20001-LG, Villeneuve-d’Ascq, France). The viability of neutrophils was above 98%. After cell counting, neutrophils were incubated at a rate of 1 × 10⁶ cells/well (in 24-well plate) or 1 × 10⁵ cells/well (in 96-well plate). The culture plates were then incubated at 37 °C under 5% CO₂.