CP was purchased from Sigma (P-9135, Sigma Aldrich). Pectinase was purchased from Shandong longke enzyme Co Ltd. His-tagged recombinant human TRAIL (rhTRAIL) was produced and purified as described previously (54 (link)). Dulbecco's Modified Eagle Medium (DMEM) (12100-046), Nutrient Mixture F-12 medium (DMEM/F-12) (12400-024), Iscove's Modified Dulbecco's Medium (IMDM) (12200-036) and fetal bovine serum (FBS) (16000-044) were obtained from Gibco. Penicillin/streptomycin (MA0110), Necrostatin-1 (MB5067), Ferrostatin-1 (MB4718), and CRID3 (MB3021) were obtained from meilunbio. U0126 (S1901), SP600125 (S1876), NAC (S0077), GSH (S0073), and Z-VAD-FMK (C1202) were purchased from Beyotime Biotechnology. 3-NPA (N106587) and TTFA (T104976) were obtained from Aladdin. All of the other reagents were of analytical grade or better. The specific antibodies used in this study are described in Table S1 .
Nutrient mixture f 12 dmem f12 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Nutrient Mixture F-12 (DMEM/F12) medium is a cell culture medium commonly used for the growth and maintenance of a variety of cell types. It is a balanced salt solution that provides the necessary nutrients, vitamins, and other components required for cell survival and proliferation in vitro.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Z vad fmk, by Beyotime (1 mentions)
Iscove s modified dulbecco s medium imdm, by Thermo Fisher Scientific (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Matrigel extracellular matrix, by Corning (1 mentions)
Y 27632, by Merck Group (1 mentions)
Rnalater rna stabilization reagent, by Qiagen (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Merck Group (1 mentions)
Al2o3 nps, by Merck Group (1 mentions)
5 5 dithio bis 2 nitrobenzoic acid dtnb mtt, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Selenium, by Merck Group (1 mentions)
11 protocols using nutrient mixture f 12 dmem f12 medium
1
Purification and Evaluation of rhTRAIL
2
IPEC-J2 Cell Cycle and Apoptosis Analysis
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Experimental design IPEC-J2s were selected as cell model. The experiment was randomly divided into three treatment groups (0.0, 0.5, or 2.0 mg/mL SBA), each treatment group with three repetitions. The treatment time was 24 h. The main indexes measured in this experiment included the percentage of the cells at different cell cycle phases, cell apoptosis rates, the protein expressions of cell cycle and apoptosis regulated proteins, the activity of caspase-3 (Casp-3) and caspase-9 (Casp-9), and the whole-cell quantitative proteome.
Cell culture IPEC-J2s were cultured in Dulbecco's Modi ed Eagle Media: Nutrient Mixture F-12 medium (DMEM/F12) (Gibco, Carlsbad, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillinstreptomycin (Sigma, USA), incubated at 37 °C and in an atmosphere of 5% CO 2 . The medium was refreshed every 2 d and the cells were sub-cultured with 0.05 % trypsin (Gibco, USA).
Cell culture IPEC-J2s were cultured in Dulbecco's Modi ed Eagle Media: Nutrient Mixture F-12 medium (DMEM/F12) (Gibco, Carlsbad, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillinstreptomycin (Sigma, USA), incubated at 37 °C and in an atmosphere of 5% CO 2 . The medium was refreshed every 2 d and the cells were sub-cultured with 0.05 % trypsin (Gibco, USA).
3
Human Tumor Tissue Digestion and Culture
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Human tissue was collected with informed consent from all patients, and all studies were approved by the Institutional Review Boards at the University of Wisconsin-Madison and the Medical College of Wisconsin (IRB# 2018-1104). Surgically resected tissue was placed in cold chelation buffer on ice for 1 h. Tissues were washed with phosphate buffered saline (PBS) and digested at 37°C in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM/F-12) medium (Invitrogen) containing 1 mg/mL collagenase (Sigma), 0.125 mg/mL dispase (Invitrogen), 10% fetal bovine serum (FBS) (Gibco), and 1% pen-strep (Gibco) for 2–3 h with intermittent shaking. The resulting cell macro-suspension was rinsed in cold PBS, re-suspended in a 1:1 mixture of DMEM/F-12 media and Matrigel extracellular matrix (Corning), plated in 50 μl droplets, and allowed to solidify at 37°C, 5% CO2 in a cell incubator. Once solidified, droplets were overlaid with DMEM/F-12 supplemented with 7% FBS, 20 μM Y-27632 (Sigma), 50 ng/ml epidermal growth factor (EGF) (Invitrogen), R-Spondin (RSPO)-conditioned medium (homemade) and 1% penicillin-streptomycin (Gibco). FBS, Y-27632, and RSPO-conditioned medium were removed from cultures if fibroblasts were out-growing tumor cells.
4
Breast Cancer Specimen Isolation and Preservation
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Infiltrating carcinoma specimens were collected from patients with breast cancer during
surgery at the Hospital of Gynecology and Obstetrics of the Mexican Institute of Social
Security (IMSS). Immediately after surgery, to confirm its tumorous nature and to avoid
contamination, the specimen was dissected and evaluated by a pathologist. One small piece
of tissue was collected in cold serum-free Dulbecco's Modified Eagle Medium: Nutrient
Mixture F-12 (DMEM/F12) medium (Invitrogen, Grand Island, New York) and transported at 4°C
to the organotypic culture laboratory for its immediate processing; another piece of
tissue was stored in RNAlater RNA Stabilization Reagent (Qiagen, Hilden, Germany) at
−80°C.
surgery at the Hospital of Gynecology and Obstetrics of the Mexican Institute of Social
Security (IMSS). Immediately after surgery, to confirm its tumorous nature and to avoid
contamination, the specimen was dissected and evaluated by a pathologist. One small piece
of tissue was collected in cold serum-free Dulbecco's Modified Eagle Medium: Nutrient
Mixture F-12 (DMEM/F12) medium (Invitrogen, Grand Island, New York) and transported at 4°C
to the organotypic culture laboratory for its immediate processing; another piece of
tissue was stored in RNAlater RNA Stabilization Reagent (Qiagen, Hilden, Germany) at
−80°C.
5
Nanoparticle-mediated Antioxidant Assays
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Al2O3NPs (product number 642991 and average particle size <30–60 nm), GSH, 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), MTT, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and propidium iodide were obtained from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS), penicillin–streptomycin, and Dulbecco’s Modified Eagle’s Medium: nutrient mixture F-12 (DMEM/F-12 medium) were purchased from Invitrogen Co (Carlsbad, CA, USA). All other chemicals were purchased from commercial sources.
6
Neuronal Culture Preparation and Immunostaining
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Dulbecco’s modified Eagle’s medium: nutrient mixture F12 (DMEM/F12) medium (Invitrogen, Bleiswijk, the Netherlands; 11330-032), glial cell line-derived neurotrophic factor (GDNF, R&D Systems Europe, Abingdon, UK; 512-GF), fetal calf serum (FCS, Bodinco BV, Alkmaar, the Netherlands), IFN-γ (Millipore, Amsterdam, the Netherlands; IF005), Penicillin/Streptomycin (Sigma Aldrich, Zwijndrecht, the Netherlands; P0781), Neurobasal A medium (Invitrogen; 10888-022), B27 supplement (Invitrogen; 17504-044), glutamine (Invitrogen; 25030-032), Trypsin/EDTA (Gibco, Bleiswijk, the Netherlands; 25300-062), selenium (Sigma Aldrich; S5261), putrescine (Sigma Aldrich; P7505), progesterone (Sigma Aldrich; P6149), insulin (Sigma Aldrich; I6634), transferrin (Sigma Aldrich; T5391), fetuin (Sigma Aldrich; F3385), bovine serum albumin (Sigma Aldrich; A8806). Antibodies: anti-PGP9.5 (Millipore; AB5925), anti-Tubulin (Covance, Rotterdam, the Netherlands; MRB-435P), anti-HuD (Millipore; AB5971), anti-peripherin (Millipore; AB1530). Secondary antibody: Alexa fluor 594 conjugated (LifeTechnologies, Bleiswijk, the Netherlands; A-21207).
7
HaCaT Cell Culture Protocol
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HaCaT cells were grown in Dulbecco's modified Eagle medium containing Nutrient Mixture F-12 (DMEM/F-12) medium (GIBCO-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U mL-1 penicillin, 0.1 mg mL-1 streptomycin, 0.25 µg mL-1 amphotericin B, and 10 µg mL-1 hydrocortisone. Cells were cultured at 37℃ in a humidified atmosphere containing 5% CO2.
8
Cultivation of Porcine Intestinal Cells
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IPEC-J2 (Porcine intestinal columnar epithelial cell line) was cultivated by applying standard cell culture techniques at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12) medium (Gibco, Waltham, MA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) penicillin-streptomycin (Sigma, St. Louis, MO, USA). The cell culture medium was exchanged every 2 days.
9
Isolation and Culture of Rat Adipose-Derived Cells
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All animal experiments in this study were conducted with the approval of the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., Ltd. All animals were housed in an environmentally (temperature and humidity) controlled room with a 12-h light or dark cycle and free access to laboratory chow and water. Perirenal adipose tissues obtained from eight-week-old male SD rats were washed 3 times in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) medium (Gibco BRL Life Technologies Inc., USA) with 1% penicillin/streptomycin (Hyclone, CA, USA) and then transferred to a petri dish. After the removal of blood vessels and fascial tissues, adipose tissues were cut into pieces (1–2 mm3) and digested at 37 °C in DMEM/F12 medium containing 0.1% collagenase I (Solarbio Life Science, China) for 1 h. The tissue precipitation was collected and resuspended in erythrocyte lysate (Solarbio Life Science, China) for 5 min at room temperature. After filtration through 100 μm nylon filter mesh (BD Falcon) and centrifugation, 1 × 106 cells were plated on a 10-cm dish with complete DMEM/F12 medium (supplemented with 10% fetal bovine serum (FBS) (Gibco BRL Life Technologies Inc., USA) and 1% penicillin/streptomycin).
10
Culturing Mouse AT II Epithelial Cells
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The MLE-12 cell line [33 (link)], a mouse AT II epithelial cell line, was obtained from the American Tissue Type Culture Collection. Cells (passage number 7–25) were maintained in advanced Dulbecco’s Modified Eagle Medium: Nutrient mixture F-12 (DMEM/F12) medium (Gibco BRL) supplemented with 1 % HEPES (Gibco BRL), 1 % insulin-transferrin-sodium selenite (Sigma-Aldrich), 1 % L-glutamine, 1 % penicillin/streptomycin, 10 nM hydrocortisone (Sigma-Aldrich) and 10 nM β-estradiol (Sigma-Aldrich). For experimental cultures, cells were seeded at a density of 0.5 x 106 cells/insert on transparent BD Falcon™ cell culture inserts (surface area of 0.9 cm2, pores with 3.0 μm diameter, PET membranes for 12-well plates). The cell culture inserts were pretreated with 45 μL Matrigel coating solution, containing 3.36 mg/mL BD Matrigel™ basement membrane matrix growth factor reduced (BD Biosciences) in Dulbecco’s modified Eagle Medium (DMEM) medium (Gibco). Inserts were placed in BD Falcon™ tissue culture plates (12-well plates) with 1 mL medium in the upper and 2 mL in the lower chamber. The cells were kept at 37 °C in 5 % CO2 humidified atmosphere for 3.5 days.
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