The culture supernatant of HepG38.7-Tet cells14 (link) was concentrated using Amicon Ultra-15 Centrifugal Filter Devices (MILLIPORE, Darmstadt, Germany) and the resulting HBV sample (HBV DNA copies 2 × 108/ml) was used as an inoculum for infection assays. HepG2-hNTCP-C4 cells cultured in a 24-well collagen-coated plate were transfected with pcDNA-F-PUF60 and then inoculated with the HBV sample (50 µl) in DMEM containing 4% polyethylene glycol (PEG) 8000 (Promega) after 12 h of transfection. The cells were washed 3 times with PBS after 24 h of infection and then subjected to RT-qPCR after 96 h of further culture.
Dulbecco modified eagle medium (dmem)
Manufactured by Promega
Sourced in United States
DMEM (Dulbecco's Modified Eagle's Medium) is a widely used cell culture medium that provides essential nutrients and growth factors to support the growth and maintenance of a variety of cell types in vitro. It is a complex mixture of amino acids, vitamins, salts, and other components designed to create an optimal environment for cell proliferation and survival.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Promega (3 mentions)
Fugene hd, by Promega (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Agarose, by Promega (2 mentions)
Amicon ultra 15 centrifugal filter device, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Microplate luminometer, by PerkinElmer (1 mentions)
Bright glo luciferase assay system kit, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Euroclone (1 mentions)
Wnt3a, by Thermo Fisher Scientific (1 mentions)
R spondin 1, by Sino Biological (1 mentions)
Fugene hd reagent, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Multimode plate reader, by Agilent Technologies (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Streptomycin sulfate, by Thermo Fisher Scientific (1 mentions)
Nthy ori 3 1, by American Type Culture Collection (1 mentions)
Powerplex 16 str genotyping, by Promega (1 mentions)
21 protocols using dulbecco modified eagle medium (dmem)
1
HepG2-hNTCP-C4 Cell HBV Infection Assay
2
Culturing Thyroid Cancer Cell Lines
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K1 (papillary TC), SW579 (squamous TC), and 8505C (anaplastic TC) cell lines (Chinese Academy of Sciences, Shanghai, China) were used in our study. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Promega, Madison, WI, U.S.A.) containing 15% FBS (HyClone, Logan, Utah, U.S.A.) and 1% streptomycin at 37°C with 95% relative humidity and 5% CO2. Cells with 80% adherence were used for subculturing. Cells were then rinsed twice with PBS and digested with trypsin (Gibco Company, Grand Island, NY, U.S.A.). The trypsin was removed when the intercellular space was enlarged. Cells were routinely passaged without suspension cells in the above-mentioned culture medium.
3
Caco-2 hACE2 Cell-Based Pseudovirus Neutralization Assay
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Human Caco-2 hACE2 cells, stably expressing the hACE2 receptor, were grown at 37°C and 5% CO2, in DMEM (Euroclone) supplemented with 10% FCS, 2 mM glutamine, and antibiotics. Generation of Caco-2 hACE2 cells has been described previously.31 (link) Caco-2 hACE2 cells were plated at a density of 3 × 104 cells/well in white, clear-bottom 96-well plates (Costar). After 24 h, 100 μL DMEM containing 5% FCS and 8 μg/mL polybrene were added to each well, and cells were incubated for 30 min. Serial (1:3) dilutions (range: from 1 μg/mL to 0.004 μg/mL final concentration) of scFvs were prepared in DMEM containing 5% FCS. Each dilution was tested in duplicate on each plate. Aliquots (60 μL) of the LV suspension were added to an equal amount of each antibody dilution (60 μL) and incubated for 1 h at 37°C. Pseudovirus:scFv mixtures (50 μL) were then added to Caco-2 hACE2 cells after medium removal. An additional 50 μL of DMEM containing 5% FCS was added to each well after 24 h. Luciferase activity (relative luciferase units [RLU]) was detected at 72 h post-infection by using Bright-Glo Luciferase Assay System Kit (Promega) in a Microplate Luminometer (Wallac-PerkinElmer).
4
TCF-luciferase Assay for Wnt Signaling
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The TCF-luciferase assay was performed as previously described [18 (link)]. Briefly, 293T cells were cultured in DMEM (Gibco Laboratories) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), transfected with pTOP Flash or pFOP Flash plasmid together with pRL-TK plasmid using Fugene HD reagent (all from Promega, Madison, WI, USA) for 6 h and then cultured in DMEM with 10% FBS overnight. Cells were starved in serum-free DMEM for 8 h and subsequently treated with different combinations and doses of Wnt3a (Peprotech, Rocky Hill, NJ, USA), R-spondin1 (Sino Biological Inc., Beijing, China), and R-spondin1-Fc fusion protein for 18 h. Luciferase activity was determined using a Dual Luciferase Reporter Assay Kit (Promega) in a BioTek multi-mode plate reader (BioTek Instruments Inc., Winooski, VT, USA) following the manufacturers’ instructions. Relative TCF-luciferase activity was calculated as (TOP-activity/TK-activity)/(FOP-activity/TK-activity). For statistical analysis, each treatment was duplicated, and the data were shown as the means ± SEM.
5
Thyroid Cancer Cell Culture Protocol
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K1 (papillary TC), SW579 (squamous TC), and 8505C (anaplastic TC) cell lines were purchased from the Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Promega, Madison, WI) containing 15% FBS (HyClone, Logan, UT) and 100 U·mL−1 streptomycin sulfate (Invitrogen-Gibco). The human thyroid follicular cell line Nthy-ori3-1 was purchased from ATCC (Manassas, VA, USA) and was incubated in RPMI Media1640 (Invitrogen-Gibco) containing 10% FBS, 100 U·mL−1 penicillin G sodium salt and 100 U·mL−1 streptomycin sulfate. The above cells were all incubated at 5% CO2 in an incubator with 95% relative humidity at 37°C. When cells reached 80% confluency, sub-culturing was conducted. After that, cells were washed 2 times with PBS and digested with trypsin. The trypsin was discarded when the intercellular space was enlarged. Cells were passaged without suspension in the culture medium.
6
Generation and Characterization of AXIN1-repaired Cell Lines
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HEK293T (RRID:CVCL_0063), JHH7 (RRID:CVCL_2805), and SNU449 AXIN1-repaired cells (clone 20, RRID:CVCL_0454) were cultured in DMEM (Gibco) supplemented with 10% (v/v) FBS (Gibco). SNU449 AXIN1-repaired cells were generated using CRISPR-Cas9 genome editing, as previously described (12 (link)). Cells were cultured in a humidified incubator maintained at 37°C with 5% CO2. All cell lines tested negative for Mycoplasma based on the real-time PCR method at Eurofins GATC-Biotech. Identity of all cell lines and clones thereof was confirmed by the Erasmus Molecular Diagnostics Department using Powerplex-16 STR genotyping (Promega).
For the preparation of conditioned medium, L-Control [L-M(TK-), RRID:CVCL_4536] and L-Wnt3A (RRID:CVCL_0635) cells were cultured in complete DMEM medium, followed by collection and filtration of medium according to the standard procedures.
For the preparation of conditioned medium, L-Control [L-M(TK-), RRID:CVCL_4536] and L-Wnt3A (RRID:CVCL_0635) cells were cultured in complete DMEM medium, followed by collection and filtration of medium according to the standard procedures.
7
Pharmacological Evaluation of Psychoactive Compounds
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MG132 from the Peptide Institute; mastoparan from Wako; Z-VAD-fmk from Promega; and DMEM with or without 4.5 g/l (25 mM) glucose and protease inhibitor cocktail from Nacalai Tesque. Olanzapine and risperidone were purchased from Toronto Research Chemicals. Various antibodies were obtained as described in Key Resource Table.
8
Evaluating Peptide-PNA Conjugates for Cellular Delivery
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PPT/HeLa cells were seeded (9.5×103 cells/well) in 96-well plates and grown overnight. Cells were washed with PBS (Gibco Life Technologies) and then incubated with PNAs or peptide-PNA conjugates in the presence or absence of 150 µM chloroquine (Sigma) in 50 µL of serum-free Opti-MEM medium or in DMEM (Gibco Life Technologies) with 7% FCS for 4 h. Luciferase expression was induced by addition of 150 µL doxycycline (1 µg/mL) in DMEM medium, and then cells were cultured for another 24 h. For cell viability assays, cells were treated in the same way, and viability was evaluated with the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer’s instructions.
9
APN/CD13 Overexpression in B16-F1 Melanoma
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HT1080, B16-F1 melanoma, H1299 and A549 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). PC14 cells were obtained from Immuno-Biological Laboratories (Gunma, Japan). HT1080, B16-F1, and A549 cells were cultured in DMEM (Gibco, Grand Island, NY, USA), and H1299 and PC14 cells were cultured in RPMI 1640 Medium (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were incubated at 37°C in an atmosphere containing 5% CO2.
A retroviral vector (pDON-5neoCD13; Takara Bio, Shiga, Japan) containing APN/CD13 cDNA and a control pDON-5neo vector were transfected into B16-F1 melanoma cells according to the manufacturer’s protocol. Transfectants resistant to 1 mg/mL G-418 (Promega, Madison, WI, USA) were selected and maintained in DMEM with 10% FBS and 1 mg/mL G-418. From these B16-F1 transfectants, one cell line that expressed high levels of APN/CD13 (APN-B16 cells) and another cell line that did not express APN/CD13 (control-B16 cells) were selected. The expression levels of APN/CD13 were determined by flow cytometry and real-time PCR.
A retroviral vector (pDON-5neoCD13; Takara Bio, Shiga, Japan) containing APN/CD13 cDNA and a control pDON-5neo vector were transfected into B16-F1 melanoma cells according to the manufacturer’s protocol. Transfectants resistant to 1 mg/mL G-418 (Promega, Madison, WI, USA) were selected and maintained in DMEM with 10% FBS and 1 mg/mL G-418. From these B16-F1 transfectants, one cell line that expressed high levels of APN/CD13 (APN-B16 cells) and another cell line that did not express APN/CD13 (control-B16 cells) were selected. The expression levels of APN/CD13 were determined by flow cytometry and real-time PCR.
10
Cell Culture Protocol for Cancer and Normal Cell Lines
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The human GBM, cancer breast (MCF-7), cervical carcinoma (HeLa), and nonmalignant human foreskin fibroblast (HF-5) cell lines were obtained from King Fahd Center for Medical Research, King Abdulaziz University, Saudi Arabia. The cells were grown in Dulbecco's modified Eagle's medium (DMEM, Promega) containing 10% fetal bovine serum (FBS, Promega) and 1% penicillin-streptomycin antibiotics (Promega) in tissue culture flasks under a humidifying atmosphere containing 5% CO2 and 95% air at 37°C. The cells were subcultured at 3-4-day interval.
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