Horse serum

PC12 cells were obtained from ATCC (CRL-1721) and cultured in RPMI 1640 (Corning, 10–040-CV) supplemented with 10% heat inactivated horse serum (Corning, 35-030-CV), 5% fetal bovine serum (Corning, 35-010-CV), 1% penicillin streptomycin (Corning, 30-002-CI) and maintained as monolayer cultures in a humidified atmosphere containing 5% CO₂ at 37 °C. Complete media was diluted appropriately to make serum deprived media containing 0.5% or 0.1% serum. T-75 flasks and 96-well plates were coated with 100 μg/mL Rat Tail Collagen I (Corning, 354249) prepared in 0.02 N acetic acid solution. The flasks and plates were coated for 4 h and washed with sterile double distilled water. The coated flasks and plates can be stored for up to a week at 4 °C. The cells obtained from ATCC were considered as passage 1 and the subsequently cultured cells as passage 2 and onward. The protocols for culturing and subculturing were used from published protocols.^{5 (link)} Etoposide (Chem-Impex International, 28435) was stored as a working stock solution of 150 μg/mL at −20 °C for up to 3 months. Etoposide is sensitive to freeze–thaw cycles and thus the number of free-thaw cycles was limited to one. The LDH assay was performed based on the manufacturer’s recommended protocol (ThermoFisher Scientific, Pierce LDH Cytotoxicity Assay Kit, 88954).

The pancreatic cancer cell line, BxPC-3 and Mia PaCa-2 were from American Type Culture Collection and grown at 37°C with 5% CO_2. The BxPC-3 cells were cultured in RPMI media (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Corning). MiaPaCa-2 cells were cultured in DMEM high glucose medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 2.5% horse serum (Corning). The cell lines were sub-cultured by enzymatic digestion with 0.25% trypsin/ 1 mM EDTA solution (Thermo Fisher Scientific) when they reached approximately 70% confluency.

PDAC cell lines (MIA PaCa-2 and PANC-1) were purchased from the American Type Culture Collection and grown at 37°C with 5% CO₂. MIA PaCa-2 cells were cultured in DMEM high-glucose medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Atlanta Biologicals), 2.5% horse serum (Corning), and 1% antibiotic/antimycotic solution (penicillin, streptomycin, amphotericin B; Hyclone). PANC-1 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution. The cell lines were sub-cultured by enzymatic digestion with 0.25% trypsin/1 mM EDTA solution (Thermo Fisher Scientific) when they reached approximately 70% confluency.