Cell culture media

Human CRC; DiFi; DKO-1; HCA-7-derived SC^{7 (link)}, CC, CC-CR; LS174T; LIM1215; MDA-MB-231 and LM2-4175 (human breast cell lines); PANC-1 (pancreatic cancer cell line); Calu-3 (lung cancer cell line); Gli36vIII (human glioblastoma cell line) and HeLa cells were cultured in DMEM medium supplemented with 10% bovine growth serum, 1% glutamine, 1% non-essential amino acids and 1% penicillin–streptomycin at 37 °C in a 5% CO₂ humidified incubator. All cell culture media were purchased from Corning Cellgro and all cell culture supplements were from Hyclone, unless stated otherwise. Primary cultures for production of EVs were initiated at passage 2 and the cells were maintained in DMEM supplemented with 2 μg ml^-1 normocin, insulin-transferrin-selenium, epidermal growth factor, hydrocortisone and T3 thyroid hormone. For the 3D cultures, the cells were cultured in type-1 collagen. Type-1 collagen was diluted at 2 mg ml⁻¹ in DMEM containing 10% FBS. Assays were set up using three collagen layers, with the middle layer containing the single-cell suspension at 5,000 cells ml⁻¹. Medium with or without reagents was added on top and changed every 2–3 d.

Primers were purchased from Integrated DNA Technologies (Coralville, IA). KOD polymerase master mix and polybrene were purchased from EMD Millipore (Darmstadt, Germany). Sequencing was performed by Retrogen Inc (San Diego, CA). Fluorescently labeled antibodies and 7-AAD for flow cytometry were purchased from Biolegend (San Diego, CA), eBioscience (San Diego, CA), or Beckman Coulter (Brea, CA). Peptides were purchased from Anaspec Inc (Fremont, CA) or Thermo Fisher Scientific (Waltham, MA). Anti-CD3 (OKT3) and anti-CD28 (CD28.2) activating antibodies were purchased from eBioscience (San Diego, CA). Short-dated Proleukin was provided by Prometheus Laboratories Inc (San Diego, CA) through an investigator-initiated trial program. All other cytokines were purchased from Peprotech, Inc. (Rocky Hill, NJ). Concanavalin A was purchased from Sigma-Aldrich (St. Louis, MO). RetroNectin was purchased from Clontech Laboratories (Mountain View, CA). BioT transfection reagent was purchased from Bioland Scientific (Paramount, CA). Cell culture media, antibiotics, and fetal bovine serum were purchased from Corning (Corning, NY). Human AB serum was purchased from Omega Scientific (Tarzana, CA). Peptides were purchased from Anaspec (San Jose, CA).