HeLa cells were cultured in a 10 cm dish (Corning) filled with Dulbecco's Modified Eagle's Medium (DMEM) at 37 °C in the presence of 5% CO2. The cells were cultured until 80% confluent, and the cells were washed with phosphate buffered saline without Ca2+ and Mg2+ (PBS(−)) after removing DMEM. Then the HeLa cells were prepared with trypsin–EDTA treatment. After trypsin–EDTA was removed, DMEM was added to the dish, and the cell suspension was transferred to a centrifuge tube. DMEM was prepared as follows: Dulbecco's modified Eagle's medium (D5796, Sigma) was mixed with Fetal bovine serum (10 vol%) (10437-028, Gibco) and Penicillin G–Streptomycin sulfate (5 vol%) (15070-063, Gibco). PBS(−) containing NaCl (8.00 g L−1), KCl (0.20 g L−1), Na2HPO4 (1.15 g L−1), and KH2PO4 (0.20 g L−1) was used for the experiments after autoclaving at 120 °C for 20 min.
Penicillin g streptomycin sulfate
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Penicillin G-streptomycin sulfate is a commonly used antibiotic solution for cell culture applications. It provides antimicrobial activity against a broad spectrum of bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (4 mentions)
6 well culture plates, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Calcium chloride cacl2, by Maclin Biochemical Technology (2 mentions)
Pmd2 g, by Addgene (2 mentions)
Pcdh cmv mcs ef1 copgfp, by System Biosciences (2 mentions)
10 cm dish, by Corning (1 mentions)
D5796, by Merck Group (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Human recombinant bfgf, by Thermo Fisher Scientific (1 mentions)
Bmp 2, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Lonza (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
14 protocols using penicillin g streptomycin sulfate
1
HeLa Cell Culturing and Preparation
2
Cell Line Characterization and Culture
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Human lung cancer cells H292, esophageal squamous cell carcinoma cells KYSE150, head and neck squamous carcinoma cells FaDu, lung adenocarcinoma cells H1299/A549, and human embryonic kidney cells HEK293T were obtained from the American Type Culture Collection (ATCC). H292 and KYSE150 cells were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS (Hyclone, Logan, UT, USA). FaDu, H1299, A549, and HEK293T cells were grown in DMEM medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS (Hyclone, Logan, UT, USA). All cells were grown in the medium containing 1% penicillin G/streptomycin sulfate (GIBCO, Rockville, MD, USA) and were incubated at 37 °C in a humidified incubator under 5% CO2. Authentication of cells was verified by short tandem repeat DNA profiling.
3
Growth Factor Inhibition Assay
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Dulbecco's modified Eagle's medium (DMEM) was purchased from Lonza (Walkersville, MD, USA). Penicillin G–streptomycin sulfate and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Recombinant human bFGF, TGFβ-1, EGF, and BMP2 were purchased from Peprotech (Rocky Hill, NJ, USA), and inhibitors of FGF (SU5402), TGFβ-1 (SB505124), and EGF (PD153035) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). These inhibitors were prepared in dimethyl sulfoxide (DMSO) and were used in growth factor inhibition assay.
4
Oxidative stress and cellular senescence
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Fetal bovine serum (FBS), α-minimum essential medium (αMEM), trypsin-EDTA, phosphate-buffered saline (PBS) and penicillin G-streptomycin sulfate were purchased from Gibco Life Technologies (Carlsbad, CA, USA). 2,7-dichlorodihydro fluoresceindiacetate (DCFH-DA) fom Sigma-Aldrich (St. Louis, MO, USA). Transwells (6-well millicell Hanging Cell Culture Inserts, 0.4 µm, PET) and 6-well culture plates were purchased from Millipore Corp. (Billerica, MA, USA). The goat anti rabbit IgG, phosphor-p44/42 MAPK rabbit mAb (p-ERK1/2), JNK MAPK rabbit mAb, p53 rabbit mAb, SIRT1 rabbit mAb and Senescence β-Galactosidase Staining kit were purchased from Cell Signaling Technology, Inc. (3 Trask Lane; Danvers, MA, USA). Penicillin and streptomycin from Gibco Life Technologies. Other reagents used were of the highest commercial grade available.
5
Maintenance of Gastric and Endothelial Cell Lines
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Human AGS (ATCC®CRL-1739TM), AGS-Ecad (Oliveira et al., 2009 (link)), Kato-III (ATCC®HTB-103TM), MKN28 (JCRB0253TM) were maintained in RPMI 1640 medium with GlutaMAXTM (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, United States) supplemented with 10% fetal bovine serum (FBS, HyCloneTM, GE Healthcare Life Sciences, Logan, UT, United States), 200 IU/mL penicillin G-200 μg/mL streptomycin sulfate (Invitrogen) at 37°C under 5% CO2 humidified atmosphere. Human umbilical vein endothelial cells (HUVECs; HUV-EC-C, ATCC®CRL1730TM) were maintained in Medium 199 (M199) with Earle’s salts, stable glutamine, and 25 mM HEPES buffer (Biowest, Nuaillé, France) supplemented with 10% FBS, 100 IU-100 μg/mL penicillin G-streptomycin sulfate (Gibco), 100 μg/mL Heparin (Sigma–Aldrich, St. Louis, MO, United States), and 30 μg/mL Endothelial Mitogen (ECGS) (BioMedical Technologies, Inc., Stoughton, MA, United States), in gelatin-coated (Sigma–Aldrich, St. Louis, MO, United States) tissue-culture Petri dishes (TPP® Plastic Products AG, Trasadingen, Switzerland), at 37°C under 5% CO2 humidified atmosphere.
6
Osteogenic Differentiation of Cells
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Fetal bovine serum (FBS), α-minimum essential medium (αMEM), trypsin-EDTA, phosphate-buffered saline (PBS) and penicillin G-streptomycin sulfate were purchased from Gibco® Life Technologies. U0126 (ERK1/2 inhibitor), 2,7-dichlorodihydrofluoresceindiacetate (DCFH-DA), β-glycerophosphate, ascorbic acid, and dexamethasone were purchased from Sigma–Aldrich (USA). Transwells (6-Well Millicell Hanging Cell Culture Inserts, 0.4 μm, PET) and 6-well culture plates were purchased from Millipore® (USA). The rabbit anti-phospho-Runx2 (Ser451) was purchased from BIOSS Bioscience (China).The Cell-Light™ EdU Apollo®488 In Vitro Imaging Kit was purchased from RiboBio (China).The goat anti-rabbit IgG, phospho-p44/42 MAPK rabbit mAb(p-ERK1/2), p44/42 MAPK (ERK1/2) rabbit mAb, and Runx2 (D1L7F) rabbit mAb were purchased from Cell Signaling Technology (USA).The protein assay kit, RIPA buffer, and DAPI were purchased from Beyotime (China). The alkaline phosphatase (ALP) assay kit and bicinchoninic acid (BCA) assay kit were purchased from the Jiancheng Corp (China).Other reagents used were of the highest commercial grade available.
7
Sodium Alginate-based Drug Delivery System
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CMC-Na (average Mw ≈ 10 kDa, degree of substitution = 0.7), minocycline hydrochloride (MH), and malachite green (MG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Na-Alg (40–90 mPa s in 1% aqueous solution, M/G ratio = 2.05, Mw = 16–34 kDa) was purchased from Fisher Scientific Co. (Pittsburgh, PA). Calcium chloride (CaCl2) was purchased from Macklin (Shanghai, China). Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island), fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials Co., China) and penicillin G-streptomycin sulfate (Life Technologies Corporation, USA) were adopted as the cell culture medium. Trypsin-EDTA (0.25% trypsin-EDTA) was purchased from Invitrogen (Carlsbad, CA, USA).
8
Biopolymer-based Tissue Engineering Scaffold
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Minocycline hydrochloride (MH), tetracycline hydrochloride (TH), and calcium chloride (CaCl2) were purchased from Macklin Chemical Co., Ltd. (Shanghai, China). The sodium salt of algal alginate extracted from brown algae Sargassum cristaefolium was obtained from Sigma-Aldrich (St. Louis, MO, USA). Al2O3 was obtained from Acros Organics (New Jersey, USA). Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, USA), penicillin G-streptomycin sulfate (Life Technologies Corporation, Chicago, USA), and fetal bovine serum (FBS, Hangzhou Sijiqing Biological Engineering Materials Co., Hangzhou, China) were used as the cell culture medium. Trypsin–EDTA (0.25% Trypsin–EDTA) was obtained from Invitrogen (Carlsbad, CA, USA).
9
Calcium-Alginate Hydrogel Cell Culture
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Calcium chloride (CaCl2) and sodium alginate (Na-Alg, Mw ≈ 20–50 kDa) were purchased from Macklin (Shanghai, China). THSG and carboxymethylcellulose sodium (CMC-Na, average Mw ≈ 10 kDa, degree of substitution = 0.7) were obtained from Sigma-Aldrich (St. Louis, MS, USA). Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, USA), fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) and penicillin G-streptomycin sulfate (Life Technologies Corporation, USA) were used as the cell culture medium. Trypsin-ethylenediaminetetraacetic acid (EDTA) (0.25% trypsin-EDTA) was purchased from Invitrogen (Grand Island, NY, USA).
10
Pluronic F127-Based Hydrogel Fabrication
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Pluronic F127 (F127) (PEG99-PPG65-PEG99, Sigma-Aldrich) was vacuum dehydrated for 4 h prior to use. The fresh TMC was obtained from Guangdong Huizhou Huayang medical device company and dried overnight in a vacuum oven prior to use. Tetramethylethylenediamine (TMEDA, 98% w/w) was purchased from J & K and used without further purification.
Tetrahydrofuran (THF, A.R), anhydrous ether, ethyl acetate and various other chemicals were purchased from Guangzhou Chemical Reagent Factory. The THF was dried via refluxing over anhydrous calcium chloride and then distilled in the presence of nitrogen protection and sodium. The other chemicals were of reagent grade and used as received.
MMC (Roche Diagnostics GmbH) was dissolved in sterile water (AquaPro; Microgen, West Caldwell, NJ). Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island,) supplemented with 10% Fetal Bovine Serum (FBS, Hangzhou Sijiqing Biological Engineering Materials Co, CHN) and Penicillin G-streptomycin sulfate (Life Technologies Corporation. USA) was used as the cell culture medium. The LDH was analyzed using the Cytotoxicity Detection Kit PLUS (Roche Diagnostics GmbH, Germany).
Tetrahydrofuran (THF, A.R), anhydrous ether, ethyl acetate and various other chemicals were purchased from Guangzhou Chemical Reagent Factory. The THF was dried via refluxing over anhydrous calcium chloride and then distilled in the presence of nitrogen protection and sodium. The other chemicals were of reagent grade and used as received.
MMC (Roche Diagnostics GmbH) was dissolved in sterile water (AquaPro; Microgen, West Caldwell, NJ). Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island,) supplemented with 10% Fetal Bovine Serum (FBS, Hangzhou Sijiqing Biological Engineering Materials Co, CHN) and Penicillin G-streptomycin sulfate (Life Technologies Corporation. USA) was used as the cell culture medium. The LDH was analyzed using the Cytotoxicity Detection Kit PLUS (Roche Diagnostics GmbH, Germany).
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