Type I-like RAEC were serum-deprived with 20 mM Hepes supplemented with DMEM (CO2 free buffering system) for 2 h, and subjected to biaxial cyclic stretch (∆SA 25% or ΔSA 37%) at 37 °C at a frequency of 0.25 Hz (15 cycles/min). Cells were stretched for 10 min (phosphorylation experiments), 1 h (HER ligand secretion experiments), or 6 h (gene expression and miRNA transfection experiments). In some experiments, cells were treated for 2 h prior to and during stretch with either the ADAM17 inhibitor TAPI2 (50 µM; Calbiochem, Burlington, MA, USA), competitive anti-HER3 antibody to prevent ligand binding (10 µg antibody/mL DMEM; Abcam, Cambridge, MA, USA), the HER2 tyrosine kinase inhibitor AG825 (50 µM; Calbiochem), or vehicle control (DMSO).
Dulbecco modified eagle medium (dmem)
Manufactured by Abcam
Sourced in United States, United Kingdom
DMEM (Dulbecco's Modified Eagle Medium) is a widely used cell culture medium that provides nutrients and essential components required for the growth and maintenance of various cell types. It is a formulation of salts, amino acids, vitamins, and other components that support cell metabolism and proliferation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Af6000, by Leica (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 1r1 fc, by Abcam (1 mentions)
Insulin transferrin selenium its g, by Thermo Fisher Scientific (1 mentions)
Anti il 1β neutralizing antibody, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
Sitagliptin, by Merck Group (1 mentions)
Tnf α, by Abcam (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
P akt, by Abcam (1 mentions)
Total pi3k, by Abcam (1 mentions)
P pi3k, by Abcam (1 mentions)
P mtor, by Abcam (1 mentions)
Automatic gel imaging system, by Bio-Rad (1 mentions)
9 protocols using dulbecco modified eagle medium (dmem)
1
Biaxial Stretch-Induced ADAM17 and HER Signaling
2
Recombinant HSV Production and Validation
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Recombinant HSV was produced as previously described.33 First, as HSV carrying Bcl-xL recombinant has lost its proliferative capacity, in order to help the replication of nonreplicated HSV Bcl-xL recombinant, ICP27 and ICP4 gene were constructed into plasmid. Next, Vero HSV packaging cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Abcam, Cambridge, United Kingdom) and plated in a 6-well plate at the concentration of 1 to 1.5 × 105. After the cells were incubated at 37 °C with 5% CO2 for 1 d and the cell confluency reached 90% to 95%, transfection began according to the manufacturer’s protocol. Briefly, the Bcl-xL recombinant overexpression vector, which expressed the ICP27 and ICP4 proteins required for the packaging of the virus containing the Bcl-xL ORF, was cotransfected into the Vero cell line. After 48 h of transfection, the culture medium was collected and centrifuged at 4 °C, 3,000g, and the supernatant was filtered. Lentiviral stocks were stored at −80°C for further use. Meanwhile, images were obtained using Leica AF6000 (Leica, Wetzlar, Germany) cell station at 48 h after transfection. The successful transfection of the Bcl-xL ORF lentivirus was confirmed by the enhanced red fluorescent protein (eRFP) in the Vero cells.
3
Chondrocyte Responses to IL-1β and Treatments
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All protocols were approved by the institutional review board of Seoul St. Mary’s Hospital (KC21SISI0337) and were performed in accordance with the Declaration of Helsinki. Human articular cartilage was acquired from patients for replacement arthroplasty or joint replacement surgery, and the chondrocytes were obtained from the cartilage and maintained in DMEM (WELGENE) containing 10% FBS (Corning), 100 units/mL of penicillin, and 100 mg/mL of streptomycin (Invitrogen), and cultured at 37°C in 5% CO2 humidified incubator. Human OA chondrocytes were seeded at 3×105 cells/well or 2×105 cells/well into 6-well or 12-well plates, respectively. Following 24 h of starvation with insulin-transferrin-selenium (ITS-G, Thermo Fisher, Waltham, MA, USA), cells were stimulated with 10 ng/ml IL-1β with or without various concentrations of TA (0.5, 1, or 2 μM), recombinant human IL-1R1 Fc (1 μg/ml, Abcam), or anti-IL-1β neutralizing antibody (1 μg/ml, Abcam) in serum-free DMEM for 1, 3, or 48 h. recombinant human IL-1R1 Fc or anti-IL-1β neutralizing antibody were used as a positive control and medium only group was used as a negative control. The supernatant and cells were collected for further analysis.
4
Investigating Sitagliptin's Anti-Inflammatory Effects
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HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Primary mouse hepatocytes were isolated from livers of 6–8 weeks male C57BL/6J mice and which were cultured with RPMI 1640 (Gibco, USA) supplemented with 10% FBS and 1% P/S. All cells were sustained at 37 °C with 5% CO2 in an incubator.
The inflammatory response was stimulated by two common inducers of cell inflammation—PS (5 μg/ml, dissolved in DMEM, Sigma-Aldrich, St. Louis, MO, USA) or TNFα (20 μM, dissolved in DMEM, Abcam, Cambridge, UK) in the presence or absence of different concentrations (0, 1, 10, 100, and 200 μM) of sitagliptin (dissolved in DMEM, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. In addition, recombinant hDPP4 protein (Abcam, Cambridge, UK) was used to upregulate the expression level of DPP4 in HepG2 cells.
The inflammatory response was stimulated by two common inducers of cell inflammation—PS (5 μg/ml, dissolved in DMEM, Sigma-Aldrich, St. Louis, MO, USA) or TNFα (20 μM, dissolved in DMEM, Abcam, Cambridge, UK) in the presence or absence of different concentrations (0, 1, 10, 100, and 200 μM) of sitagliptin (dissolved in DMEM, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. In addition, recombinant hDPP4 protein (Abcam, Cambridge, UK) was used to upregulate the expression level of DPP4 in HepG2 cells.
5
Hypoxia/Reoxygenation Injury in Cardiomyocytes
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The rat myocardial cell line H9c2 was purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. H9c2 cardiomyocytes were grown in DMEM (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Wisent, Montreal, Canada), 100 units/mL penicillin, and 100 units/mL streptomycin at 37°C with 5% CO2 in a humidified atmosphere. H/R conditions were produced by placing cells in a H/R chamber (1% O2, 94% N2, and 5% CO2) for a specific period of time.
CyPA preparation: CyPA (Abcam, ab86219) was dissolved in DMEM for the treatment of cells.
GSK690693 preparation: GSK690693 (MCE, HY-10249) is a novel Akt kinase inhibitor. GSK690693 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 μM before use.
GSK2795039 preparation: GSK2795039 (MCE, HY-18950) is a NADPH oxidase 2 inhibitor. GSK2795039 was dissolved in DMSO at a concentration of 10 μM before cell treatment.
CyPA preparation: CyPA (Abcam, ab86219) was dissolved in DMEM for the treatment of cells.
GSK690693 preparation: GSK690693 (MCE, HY-10249) is a novel Akt kinase inhibitor. GSK690693 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 μM before use.
GSK2795039 preparation: GSK2795039 (MCE, HY-18950) is a NADPH oxidase 2 inhibitor. GSK2795039 was dissolved in DMSO at a concentration of 10 μM before cell treatment.
6
Phosphorylation of PI3K/AKT/mTOR Pathway
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p-PI3K, p-AKT, p-mTOR and total-PI3K, AKT, mTOR were all purchased from abcam; DMEM, PBS buffer and trypsin were purchased from Hyclone Corporation, USA; Fetal bovine serum from GIBCO, USA; Tissue embedding machine (model: EG11508) and paraffin section model (model: RM2135) were purchased from Leica Company; Western Blot electrophoresis tank (DYCZ-24DN), transfer electrophoresis apparatus (DYY-7B) and thermostat circulator (WD-9412A) were purchased from Beijing Liuyi Biological Technology Co., Ltd; Automatic gel imaging system (model: 8845-S) was purchased from Bio-rad Corporation, USA; The electronic balance (model: MP200A) was purchased from Shanghai Jingke Instrument Factory; The ice making machine (model: SIM-F124) was purchased from Japan 5ANY0 Co., Ltd; Enzyme-label instrument (model: ELX800, Beijing Bio-Tek Company) and other related reagents and instruments.
7
HEK293 Cell Culture Protocol
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HEK293 cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. All cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Abcam, USA) with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin). The cell culture was carried out in humidified air at 37°C containing 5% CO2. All cultures used standardized cell culture.
8
Anti-VEGF Trap Delivery in Triple-Negative Breast Cancer
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MDA‐MB‐231 cell lines were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA) and stored according to supplier's instructions. AAV2‐VEGF‐Trap was constructed in the State Key Laboratory of Biotherapy, West China Hospital of Sichuan University. Paclitaxel was obtained from Beijing Ruikang Pharmaceutical Industry (China). AngioSense680 EX was obtained from PerkinElmer, Inc (Boston, MA, USA). DMEM and fetal bovine serum were purchased from Gibco (USA). Anti‐Ki‐67 and anti‐CD31 are rabbit polyclonal antibodies purchased from Abcam (Shanghai, China).
MDA‐MB‐231 cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin) at 37°C in a humidified 5% CO2 atmosphere.
MDA‐MB‐231 cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin) at 37°C in a humidified 5% CO2 atmosphere.
9
Synchronizing NIH-3T3 Cells for Cell Cycle Analysis
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Mouse embryonic fibroblast NIH-3T3 cells were cultured in DMEM (GIBCO, Carlsbad, CA, USA) supplemented with 10% FBS (GIBCO, Carlsbad, CA, USA) and 1% Pen/Strep (GIBCO, Carlsbad, CA, USA) at 37℃, 5% CO 2 .
To obtain G1/S synchronized NIH-3T3 cells, cells were cultured with starvation treatment (DMEM with 0.5% FBS and 1% Pen/Strep) for 48 h, followed with fresh medium containing 1% Aphidicolin (Abcam, Cambridge, MA, USA) for 18 h before collection. With this procedure, about 97% of the collected cells were G1/S cells based on FACS (Fluorescence-Activated Cell Sorting) analysis (Supplementary Figure S1A, http://www.biosciencetrends.com/action/ getSupplementalData.php?ID=168). To obtain mitotic cells, cells in culture reached 70-80% confluence were treated with colcemid (100 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) for 12 h, and mitotic cells were shaken-off and collected for mitotic chromosome purification. The purity of the collected mitotic cells was about 77% by FACS analysis (supplementary Figure S1B, http://www.biosciencetrends.com/action/ getSupplementalData.php?ID=168).
To obtain G1/S synchronized NIH-3T3 cells, cells were cultured with starvation treatment (DMEM with 0.5% FBS and 1% Pen/Strep) for 48 h, followed with fresh medium containing 1% Aphidicolin (Abcam, Cambridge, MA, USA) for 18 h before collection. With this procedure, about 97% of the collected cells were G1/S cells based on FACS (Fluorescence-Activated Cell Sorting) analysis (Supplementary Figure S1A, http://www.biosciencetrends.com/action/ getSupplementalData.php?ID=168). To obtain mitotic cells, cells in culture reached 70-80% confluence were treated with colcemid (100 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) for 12 h, and mitotic cells were shaken-off and collected for mitotic chromosome purification. The purity of the collected mitotic cells was about 77% by FACS analysis (supplementary Figure S1B, http://www.biosciencetrends.com/action/ getSupplementalData.php?ID=168).
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