Dulbecco s modified eagle media dmem

Primary rat astrocytes were obtained from Sprague Dawley rat brains. Rats were anesthetized with pentobarbital and decapitated for brain removal. Brain tissue was minced and disrupted in ice-cold Hank’s balanced salt solution with trypsin (Sigma, St. Louis, MO). Debris was removed from cells by sedimentation, and cells were recovered from the suspended phase above the settled debris. The process was repeated, discarding the first three extractions to reduce the presence of blood vessels and red blood cells. Cells were pooled and seeded in flasks with growth medium (Dulbecco’s Modified Eagle Media (DMEM) (Sigma) supplemented with 10% fetal bovine serum, 10 mM L-glutamine, 5% non-essential amino acids, and streptomycin/penicillin) and incubated at 37°C and 5% CO₂. The non-attached cells were removed after three days in culture. Cells were cultured and used in experiments once three passages were completed to assure the cultures were astrocytes (positive staining of all cells for GFAP; neither neurons nor microglia were detectable by immunoblotting for cell-specific markers MAP-2 and Iba-1, respectively).

All chemicals and reagents including custom designed primers were obtained from Sigma Chemicals (St Louis, MO) unless otherwise stated, and were of molecular biology or research grade. Rabbit polyclonal anti-cytochrome p450 2E1 antibody (anti-CYP2E1, ab28146) was obtained from Abcam (Cambridge, MA). Mouse anti-cAMP dependent Protein Kinase [Catalytic subunit] antibody (anti-PKA[C], #610981) was purchased from BD Transduction Laboratories. Rat anti-germ cell nuclear antigen antibody (anti-GCNA) was a gift from Dr. George Enders [24] (link). Secondary antibodies, Alexa Fluor 594 goat anti-rabbit immunoglobulin G (IgG) (A11012) was purchased from Invitrogen (Carlsbad, CA). Dulbecco's Modified Eagle Media (DMEM) and supplements for cell culture were obtained from Sigma and Invitrogen. Acrylamide was obtained from Sigma (≥99% purity, A9099) and glycidamide (98% purity, G615250) was obtained from Toronto Research Chemicals (North York, CA). Oligo(dT)15 primer, RNasin, dNTPs, M-MLV-Reverse Transcriptase, RQ1 DNase, GoTaq Flexi, MgCl₂ and GoTaq quantitative PCR master mix were obtained from Promega (Madison, WI). DNA repair endonucleases, formamidopyrimidine-DNA glycosylase (FPG) and 8-oxoguanine DNA glycosylase (hOGG1) were purchased from New England Biolabs Inc. (Arundel, Qld).

Sodium selenite (98%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), Tween 80, methanol, acetate buffer, tetracycline, nystatin, rhodamine 123, caspase-3 kit, Dulbecco’s modified Eagle media (DMEM), Folin-Ciocalteu reagent, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), Dulbecco’s phosphate buffered saline pH 7.4 (DPBS) and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (Bengaluru, India). The microbial culture media, including Muller Hinton Broth (MHB), Muller Hinton Agar (MHA), brain heart infusion (BHI) broth, sabouraud dextrose agar (SDA), and sabouraud dextrose broth (SDB) were obtained from HiMedia (Mumbai, India). The live/dead dual staining assay kit was purchased from Thermo Fisher Scientific (Bengaluru, India). The plastic and glassware were obtained from Nunc and Borosil, respectively (Bengaluru, India). The other chemicals used in the study were belonged to analytical grade and were obtained from Merck Millipore (Bengaluru, India).

MTT, MTT-luciferase, and human hPheo1 cell lines were used in this study [30 (link),31 (link)]. MTT and MTT-luciferase cells are rapidly growing cells derived from liver metastases of MPC cells, and MTT-luciferase cells are transfected by a luciferase plasmid [30 (link),31 (link)]. hPheo1 (obtained from University of Texas, Southwestern Medical Center, Dr. Hans Kumar Ghayee, D.O., MTA# 41611) is a progenitor cell line derived from human PHEO [53 (link)].
MTT and MTT-luciferase cells were maintained in Dulbecco’s modified eagle media (DMEM) (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (Gemini, West Sacramento, CA, USA) and penicillin/streptomycin (100 U/mL; Gemini). In MTT-luciferase cells, geneticin (750 μg/mL; Thermo Fisher Scientific, Waltham, MA, USA) was used for stable cell line selection. hPheo1 cells were maintained in RPMI (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum and penicillin/streptomycin (100 U/mL). Both cell lines were cultured at 37 °C in humidified air with 5% CO₂.
All cell lines used were routinely tested for mycoplasma using the MycoAlert^TM detection kit (Lonza, Walkersville, MD, USA). Cell authentication was performed per ATCC guidelines using morphology, growth curves, and mycoplasma testing. After thawing, cells were cultured for no longer than 3 weeks.