Penicillin streptomycin p s

Primary human SCs, obtained from the spinal nerve cells of a healthy donor, were purchased from ScienCell (cat. no. 1700, CA, United States) and maintained (maximum of 10 passages) according to manufacturer instructions described previously (21 (link)). Briefly, T-75 culture flasks were coated with 10 mg/mL poly-L-lysine (cat. no., 0413, ScienCell) and incubated overnight at 37°C. Cells were seeded at 5,000 cells/cm² on the poly-L-lysine–coated flask after washing the vessel twice with sterile milli-Q water. Cells were grown in complete SC medium (SCM, cat. no., 1701, ScienCell), supplemented with 5% fetal bovine serum (FBS, cat. no., 0025, ScienCell), 1% SC growth supplement cocktail (SCGS, cat. no., 1752, ScienCell), and 1% penicillin/streptomycin (P/S, cat. no., 0503, ScienCell) in a humidified incubator at 37°C with 5% CO₂. SCs were characterized by immunoblotting using antibodies against human SC marker proteins, SOX10, and p75 (Supplementary Figure S1).
Pancreatic ductal adenocarcinoma cells, PANC-1 and MIA PaCa-2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States) and maintained in Dulbecco modified eagle medium (cat. no., ATC302002, ATCC) supplemented with 10% (vol/vol) FBS (JRH Biosciences, St. Louis, MO, United States) and 2 mM L-glutamine in a humidified incubator at 37°C with 5% (vol/vol) CO₂.

hiPSCs were differentiated to astrocytes based on our recent protocol [10 (link)]. Fetal human cortical astrocytes were obtained from ScienCell (#1800). Cells were cultured according to suppliers’ instructions and used at passage <8 post-thawing. One million cells were thawed in complete astrocyte media (AM; ScienCell 1801). For assays, 500,000 cells/well were seeded in 6-well cell culture plates coated with 1x attachment factor (AF; Thermo S-006-100). On the next day of subculture (day 1), the media were changed to a 1:1 mixture of AM and “defined media,” and on day 2 to defined media only. Our defined media were composed of DMEM without glucose and glutamine (Thermo A14430-01) supplemented with 1% G-5 (Thermo 17503012; serum-free supplement for growth and expression of glial cells), 1% Penicillin-Streptomycin (P/S; ScienCell 0503), 10mM GlutaMax (Sigma 35050-38), 25 mM HEPES (EMD 391340), and 5.55 mM glucose (Sigma G7528) to match the AM glucose content. We used these defined media to have better control over the initial concentration of glucose within the media. Exemplary bright-field images of the cells on day 2 are shown in Figure 1b,c.

Primary HBFs and fibroblast medium (FM) were obtained from ScienCell (USA). HBFs at passages 4−8 were cultured in fibroblast medium containing 2% foetal bovine serum (FBS; ScienCell) supplemented with 1% penicillin‐streptomycin (P/S; ScienCell) and 1% fibroblast growth supplement (ScienCell). Human lung fibroblast 1 (HFL1, ATCC CCL‐153) cells, F‐12K (Kaighn's modification of Ham's F‐12 medium, ATCC 30−2004), BEAS‐2B (ATCC CRL‐9609) cells, A549 (ATCC CCL‐185) and RPMI‐1640 medium (ATCC 30−2001) were obtained from ATCC (Manassas, USA). HFL1 cells were cultured in F‐12K medium containing 10% FBS (Gibco, USA) and 1% P/S (Invitrogen, USA), whereas BEAS‐2B and A549 cells were cultured in RPMI‐1640 medium containing 10% FBS and 1% P/S. The above primary cells or cell lines were all incubated at 37°C under a humidified atmosphere containing 5% CO₂.

Human umbilical vein endothelial cells (Cat. no. 8000; ScienCell Research Laboratories, Carlsbad, CA, United States) were cultured in ECM (Cat. no. 1001; ScienCell Research Laboratories) containing 5% fetal bovine serum (FBS; cat. no. 0025; ScienCell Research Laboratories), 1% penicillin/streptomycin (PS; cat. no. 0503; ScienCell Research Laboratories), and 1% endothelial growth factor (EGF; cat. no. 1052; ScienCell Research Laboratories). A293 cells (Fenghbio, Changsha, China) were cultured in Dulbecco’s modified Eagle medium (DMEM; 4.5 g/L D-glucose, HyClone, Logan, UT, United States) containing 10% FBS and 1% PS. All cells were cultured in a humidified incubator at 37°C with 5% CO₂. ECM containing 2% FBS, 1% PS, and 1% EGF along with D-glucose (Sigma-Aldrich, St. Louis, MO, United States) was used to generate hyperglycemic conditions. D-mannitol (Sigma-Aldrich) was also used to induce a similar osmotic pressure. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States) was used to transfect cells according to the manufacturer’s protocol. NRF2 inhibitor ML385 (3 μM) and HO-1 inhibitor ZnPP (10 μM) were used to incubate cells.

HDF (ScienCell Research Laboratories, Cat. No. 2310, Carlsbad, CA, USA) used cells between passages 4 and 6. Culture media was Fibroblasts Media (FM; ScienCell Research Laboratories, Cat. No. 2301, Carlsbad, CA, USA) containing 5% Fetal Bovine Serum (FBS; ScienCell Research Laboratories, Cat. No. 0010, Carlsbad, CA, USA), 1% Penicillin/Streptomycin (P/S; ScienCell Research Laboratories, Cat. No. 0503, Carlsbad, CA, USA), and 1% Fibroblast Growth Supplement (FGS; ScienCell Research Laboratories, Cat. No. 2352, Carlsbad, CA, USA). HEM (ScienCell Research Laboratories, Cat. No. 2200, Carlsbad, CA, USA) used cells between passages 4 and 7. Culture media was Melanocytes Media (MelM; ScienCell Research Laboratories, Cat. No. 2201, Carlsbad, CA, USA) containing 1% Melanocyte Growth Supplement (MelGS; ScienCell Research Laboratories, Cat. No. 2252, Carlsbad, CA, USA), 1% P/S, 0.5% FBS. Also, 0.25% Trypsin-EDTA (Gibco, Cat. No. 15400, Grand Island, NY, USA) was used to harvest the cells when the cells reached 80–90% confluency.