Doxorubicin (DOX) and methotrexate (MTX) were obtained from Beijing Solarbio Science & Technology Co., Ltd. 2,3-Dimethylmaleic anhydride (DMMA) was sourced from Shanghai Yuanye Bio-Technology Co., Ltd. MTX-PEG was obtained from Yarebio. Chitosan (CG), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Shanghai) Trading Co. Ltd. Cell Counting Kit-8 (CCK-8) was sourced from Dojindo Laboratories. Fetal bovine serum (FBS) was sourced from Gibco Laboratories. 1% antibiotics (streptomycin 100 mg/mL and penicillin 100 U/mL), DMEM-H/F-12 medium and DMEM/HIGH Glucose medium were obtained from HyClone Laboratories. Antibodies for Beclin-1 (D40C5), LC3A/B (D3U4C), SQSTM1/p62 (D5L7G) and GAPDH (D16H11) were sourced from Cell Signaling Technology.
Dmem high glucose medium
Manufactured by Cytiva
Sourced in United States, China
DMEM high glucose medium is a cell culture medium formulated with a high concentration of glucose to support the growth and maintenance of various cell lines. It provides essential nutrients, vitamins, and salts required for cell proliferation and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Cytiva (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Penicillin, by Cytiva (2 mentions)
Streptomycin, by Cytiva (2 mentions)
Dmem h f 12 medium, by Cytiva (2 mentions)
Sorafenib, by Selleck Chemicals (2 mentions)
Doxorubicin, by Merck Group (2 mentions)
Camptothecin, by Merck Group (2 mentions)
Penicillin streptomycin, by Cytiva (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Insulin, by Thermo Fisher Scientific (2 mentions)
Mcf 7, by Thermo Fisher Scientific (2 mentions)
Mycoplasma free, by Eurofins (2 mentions)
Rpmi 1640, by Cytiva (2 mentions)
Hepg2, by LGC (2 mentions)
Hepg2, by Cytiva (2 mentions)
Bt 474, by Cytiva (2 mentions)
Css fbs, by Cytiva (2 mentions)
16 protocols using dmem high glucose medium
1
Doxorubicin and Methotrexate Nanoparticle Formulation
2
Green Ball Apple Peel Ethanol Extract
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Green ball apple peel ethanol extract (GBE) were prepared as previously reported [1, 13] . Briefly, 1 g of fruit powder was mixed with 100 mL of 10-100% ethanol and stored at 4 o C for 24 h with shaking. Ethanol extracts were filtered through Whatman No. 1 filter paper (Whatman Inc., Piscataway, NJ, USA), lyophilized in a freeze dryer (FD8518; ilShin BioBase, Yangju, Korea), and stored at -20 o C until use. GBE were tested at concentrations ranging from 100 to 500 μg/mL. Raw 264.7 macrophage cell culture The Raw 264.7 macrophages line was purchased from Korean Cell Line Research Foundation. Cells were cultured in DMEM high glucose medium (HyClone Laboratories, Inc., Logan, UT, USA) supplemented with 10% FBS (HyClone Laboratories, Inc.) and 1% penicillin-streptomycin (HyClone Laboratories, Inc.). All incubations were performed at 37 o C with 95% humidity and an atmosphere of 5% CO 2 (311, Thermo Fisher Scientific, Rockford, IL, USA). Cells were passaged after reaching 80-90% confluence. Experiments were conducted using cells that did not exceed 20 passages.
3
Investigating MDRL and miR-361 in HEK-293T and MSMC Cells
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HEK-293T cells from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose medium (Hyclone Laboratories Inc., UT, USA) supplemented with penicillin (100 units/ml, Hyclone, USA) and fetal bovine serum (FBS, 10%, Hyclone Laboratories Inc., UT, USA), at 37°C and 5% CO2 in a humidified chamber.
Mouse aortic smooth muscle cells (MSMCs) were isolated from C57BL/6J mice. Cells were maintained in DMEM containing 10% FBS and 2 mM glutamine and passaged every 3-4 days. MSMCs at Passages 2-4 were used for further study.
As shown in TableS1 , silencing RNAs (siRNAs), miRNA mimics or inhibitors, and corresponding controls were designed and synthesized by Genepharma Co., Ltd. (Suzhou, China). Oligonucleotides (50 nM) were transfected into HEK-293T cells or MSMCs using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer's instructions. Cells were harvested for qRT-PCR or western blot analysis after 48 h of transfection. MDRL cDNA sequences were subcloned, and then gene expression was amplified from the lentiviral vectors. To explore the effect of MDRL on cell function and miR-361, MSMCs and HEK-293T cells were transfected and divided into four groups: scramble siRNA (MDRL-sc), MDRL-siRNA, control adenoviral, or adenoviral MDRL.
Mouse aortic smooth muscle cells (MSMCs) were isolated from C57BL/6J mice. Cells were maintained in DMEM containing 10% FBS and 2 mM glutamine and passaged every 3-4 days. MSMCs at Passages 2-4 were used for further study.
As shown in Table
4
Preparation and Characterization of HA-based Nanosystem
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Sodium hyaluronate (HA, MW=90 kDa) was purchased from Bloomage Freda Biopharm. N-hydroxysuccinimide (NHS) and 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) were obtained from Macklin. Auraptene (AUR), mono-(6-amino-mono-6-deoxy)-β-CD (CD) and cisplatin (CDDP) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Dimethyl sulfoxide (DMSO) and 4ʹ-6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories. Fetal bovine serum (FBS) was obtained from Gibco Laboratories, and DMEM/HIGH Glucose medium, DMEM-H/F-12 medium and 1% antibiotics (penicillin 100 U/mL, and streptomycin 100 mg/mL) were purchased from HyClone Laboratories. Human breast cancer MCF-7 cells were obtained from BeNa Culture Collection (BNCC). HK-2 cells were purchased from American Type Culture Collection (ATCC).
5
Culturing HEK 293 Cells in DMEM
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Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco's Modified Eagle's Medium purchased from Sigma Aldrich Inc. and supplemented with 10% fetal bovine serum (FBS) purchased from Atlas biologicals, DMEM – high glucose medium, and antibiotic/antimycotic solution (100×) purchased from Hyclone laboratories.
6
Establishing Bioluminescent Cancer and NK Cell Lines
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The human thyroid cancer cell line CAL-62 was purchased from the American Type Culture Collection (Rockville, MD, USA). The thyroid cancer cells were maintained in DMEM/high-glucose medium (HyClone Laboratories, Inc., South Logan, UT, USA) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. The NK-92MI human NK cell line was obtained from the American Type Culture Collection. NK-92MI cells were incubated in stem cell growth medium (Cellgro, Freiburg, Germany) supplemented with 2% human serum and 100 U/ml penicillin/streptomycin. CAL-62 cells were transduced with lentivirus co-expressing the Rluc (Renilla luciferase), mcherry, and puromycin genes under the control of the CMV promoter (Genecopoeia, Rockville, MD, USA). Stable clones were selected with puromycin (Sigma-Aldrich, St. Louis, MO, USA). CAL-62 and NK-92MI cells were retrovirally transduced to express both the effluc (enhanced firefly luciferase) and Thy1.1 genes. Thy1.1-positive cells were sorted by CD90.1 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The established stable cell lines expressing the Rluc and effluc genes were referred to as the CAL-62/R, CAL-62/F, and NK/F cells.
7
NSCLC and Osteoclast Cell Lines Cultivation
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Four kinds of NSCLC cell lines A549, H1355, H1299 and H460, human bronchial epithelial cell line 16HBE, OC precursor RAW264.7 cells and human OB SaOS-2 cells were obtained from ATCC. After thawing, the cells were kept in DMEM high glucose medium (Hyclone Laboratories, San Angelo, TX, USA) containing 10% fetal bovine serum (Tianhang Biological Technology, Huzhou, China), 100 U/mL penicillin (Biosharp, China) and 0.1 mg/mL streptomycin (BioSharp, Hefei, China). The cultivation environment was 37°C and 5% CO2 (link). For the induction of RAW264.7 cells, the Transwell chambers were used. Specifically, 1.104/mL of the transfected A549 cells and 5.103/mL of the RAW264.7 cells were inoculated in the upper and lower chamber, respectively. They were co-cultured in 37°C and 5% CO2 (link) for 7 days, then the differentiation of RAW264.7 cells into OCs was detected. In addition, to elucidate the effect of OPN on bone metabolism through Notch pathway, RAW264.7 cells and SaOS-2 cells were treated with Notch pathway inhibitor LY3039478 (final concentration was 1 nM).
8
Inhibition of miR-451 in hUCMSCs
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The hUCMSCs were obtained from the First Affiliated Hospital of Anhui Medical University and maintained in Dulbecco's modified Eagle's medium (DMEM)/high glucose medium (HyClone Laboratories, Logan, UT, USA) supplemented with 10% defined fetal bovine serum (HyClone Laboratories) at 37°C in 5% CO 2 .
The HUCMSCs were transfected with a miRNA inhibitor control or a miR-451 inhibitor using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.
The HUCMSCs were transfected with a miRNA inhibitor control or a miR-451 inhibitor using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.
9
Cell Culture Protocol for Liver Cancer
10
Culturing Liver Cancer and Normal Cells
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HepG2 and PLC-PRF5 cells were obtained from American Type Culture Collection (Manassas, VA), and cultured in DMEM high glucose medium (HyClone Laboratories, Logan, UT), containing 10% fetal bovine serum and 1% antibiotic–antimycotic (Invitrogen, Grand Island, NY), at 37 °C with 5% CO2. Non-malignant human hepatocytes HH were obtained from Sciencell and cultured as recommended by the supplier. For all studies with extracellular vesicles, vesicle depleted medium was prepared by centrifuging cell-culture medium at 100,000g overnight to spin down any pre-existing vesicle content. Camptothecin and doxorubicin were obtained from Sigma-Aldrich (St. Louis, MO), and sorafenib was obtained from Selleck (Houston, TX). Compounds were dissolved in 100% DMSO (Sigma–Aldrich, St. Louis, MO) and diluted with culture media to the desired concentration with a final DMSO concentration of 0.1%. DMSO 0.1% (v/v) was used as a solvent control.
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