Dulbecco s modified eagle s medium

The prostate cancer cell lines LnCap, PC3 and DU145 were purchased from the Wuhan University Strain Preservation Center (Wuhan, China) and cultured in Ham's F12 medium or Dulbecco's modified Eagle's medium (Corning, US). These cells were supplemented with exosome-free serum Ham's F12 medium or Dulbecco's modified Eagle's medium supplemented with exosome-free serum (Corning, US), 2 ng/mL basic fibroblast growth factor (Corning, US), 2 mM glutamine (Yongjin Biotech, Guangzhou, China), and 10 units/ml penicillin/streptomycin. Cultures were maintained at 37 °C in an atmosphere containing 5% CO₂.

In order to generate autologous tumor cells for immunological analyses, the amputated limb was collected from surgery and small tumor pieces were removed and diced into 1 mm cubes. The remaining tumor mass was submitted for histopathological confirmation of the diagnosis of OSA. The tumor cubes were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium, Corning) with penicillin (300 IU/ml, Corning), streptomycin (300 ug/ml, Corning), amphotericin B (1.5ug/ml, Corning), and 20% FBS (fetal bovine serum, Sigma) at 37 °C in 6 well plates as tumor explants. The media was changed weekly until cells were 80–90% confluent. Adherent cells were passaged two to four times before cryopreservation. Cells were cryopreserved in Freezing medium (10% DMSO + complete DMEM media) by gradually cooling the cells to -80 °C. Cryopreserved cells were stored in liquid nitrogen. Cryopreserved OSA cells were thawed at 37 °C. OSA cells were cultured in complete DMEM (Dulbecco's Modified Eagle's Medium, Corning) with penicillin (100 IU/ml, Corning), streptomycin (100 ug/ml, Corning), amphotericin B (0.5ug/ml, Corning), and 20% FBS (fetal bovine serum, Sigma) at 37 °C.

RAW 264.7 (American Type Culture Collection, Rockville, MD, USA), a mouse macrophages cell line, was cultured in Dulbecco’s modified Eagle’s medium (Cellgro, VA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Invitrogen Co., Waltham, MA, USA), and 4 mM l-glutamine at 37 °C in an atmosphere of 5% CO₂.

The human pancreatic cancer cell line PANC-1 was obtained from the American Type Culture Collection (Rockville, MD). It harbors several common mutations in pancreatic cancer, including KRAS^G12D, TP53^R273H, and homozygous deletion of CDKN2A [23 (link)]. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (Cellgro, Manassas, VA) containing 10% (v/v) fetal bovine serum (Atlanta Biological, Lawrenceville, VA) in a humidified atmosphere with 5% CO₂ at 37 °C. Cells were passaged at 80 ~ 90% confluence using 0.05% trypsin with 0.53 mM EDTA (Gibco BRL, Gaithersburg, MD).

The human lung carcinoma cell line A549 was maintained in F-12K medium (Corning Cellgro) supplemented with 10% fetal bovine serum (Thermo Scientific) and antibiotics (100 U/ml penicillin G and 100 g/ml streptomycin). Madin-Darby canine kidney (MDCK) and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Corning Cellgro) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin G and 100 g/ml streptomycin). All cells were cultured at 37°C and in 5% CO₂.