Dmem high glucose hyclone medium

HEK 293 T cells (human embryonic kidney cells, ATCC, Teddington, Middlesex, UK) were seeded and grown in DMEM/high glucose Hyclone medium (GE Healthcare Life Sciences, South Logan, UT), supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum to approximately 95% confluence before transfection. Transient transfection was performed with Lipofectamine (Invitrogen, Life Technologies, Waltham, MA) according to the manufacturer’s instructions. A control well without added DNA was included. Twenty-four hours after transfection, the medium was changed to Optimem (Thermo Fisher Scientific), and cells were cultured for an additional 72 h. The media were collected, supplemented with protease inhibitors cOmplete Mini without EDTA (Roche Diagnostic, Mannheim, Germany) and centrifuged to separate supernatant and cell debris. The supernatant was concentrated 25 × to 200 µL using a 10 kDa filter (Ultra-4 Centrifugal Filter Units, Merck Millipore, Billerica, MA). The cells were diluted in RIPA buffer for protein extraction (Sigma) with cOmplete Mini without EDTA, scraped from the wells, and the dilution underwent three freeze-thaw cycles to enhance lysis.

Transfection was performed as previously described (27 (link)). Briefly, human embryonic kidney 293 cells (HEK) cells (ATCC, Teddington, Middlesex, UK) were seeded and grown in DMEM/high glucose Hyclone medium (GE Healthcare Life Sciences, South Logan, UT), supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum to approximately 95% confluence before transfection. Plasmid DNA (2 μg) was added to each well and transfection performed with Lipofectamine (Invitrogen, Life Technologies, Waltham, MA) according to the manufacturer’s instructions. Twenty-four h after transfection, the medium was changed to Optimem (Thermo Fisher Scientific) and cells were cultured for an additional 72 h. The media were collected and supplemented with protease inhibitors cOmplete Mini without EDTA (Roche Diagnostic, Mannheim, Germany) and centrifuged to remove cell debris.

Pancreatic stellate cells (PSCs, ScienCell, Carlsbad, CA, USA) were cultured in stellate cell medium supplemented with 2 v/v % fetal bovine serum (FBS), 100 U/mL penicillin/100 µg/mL streptomycin (Pen/Strep) and 1 v/v % stellate cell growth supplement (SteCGS) according to manufacturer’s instruction (all products from ScienCell). Panc-1 cancer cells (ATCC, Manassas, VA, USA) were cultured in DMEM - High Glucose HyClone medium (GE Healthcare Life Sciences, Chicago, IL, USA) containing 10 v/v % FBS, 100 U/mL penicillin/100 µg/mL streptomycin (Thermofisher Scientific) and 2 mM L-glutamine (Thermofisher Scientific). The cells were maintained at 37 °C in a humidified 5% CO₂ atmosphere and passed at 80% confluence. Passing of cells was performed as follows: The cells were washed twice with warm Dulbecco’s phosphate buffered saline (DPBS) (Lonza, Basel, Switzerland) before addition of trypsin/ethylenediaminetetraacetic acid (EDTA) (Thermofisher Scientific) and incubation at 37 °C. The trypsin/EDTA mix was neutralized by using 10× cell culture medium before being transported to a sterile Falcon tube and counted using a hemocytometer (Buerker-Tuerk, Brand GMBH, Wertheim, Germany). PSCs were used from a passage of 4–10.