A2780 and SKOV3 cell lines were purchased from the European Collection of Cell Cultures (ECACC, London, UK) and Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) respectively. Each cell line was seeded in a 96-well plate at an appropriate cell number (3000 cells/100 μL in each well). After 24 h, butein was added to the plates at each of the following concentrations, 0, 3.125, 6.25, 12.5, 25, and 50 μM, in triplicate in the presence of serum medium. After 48 h, 100 μL of N, N-dimethylformamide (Sigma-Aldrich, St. Louis, MO, USA) solubilizing solution was added to each well and incubated for 4 h. The solution was then aspirated, and 100 μL of DMSO was added for cell lysis. The spectrophotometric absorbance was measured at 540 nm. Half of the maximum inhibitory concentration (IC50) was determined using GraphPad Prism software (version 7.0).
Skov3
Manufactured by European Collection of Cell Cultures
Sourced in United Kingdom
The SKOV3 is a cell line derived from a human ovarian adenocarcinoma. It is a commonly used in vitro model for ovarian cancer research.
Automatically generated - may contain errors
Lab products found in correlation
A2780, by European Collection of Cell Cultures (5 mentions)
Skov3, by Thermo Fisher Scientific (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Mia paca 2, by American Type Culture Collection (2 mentions)
Mccoy s 5a medium modified, by Lonza (2 mentions)
Aspc 1, by American Type Culture Collection (2 mentions)
Iscove modified dulbecco media (imdm), by Lonza (2 mentions)
Tissue culture flasks, by Sarstedt (2 mentions)
Streptomycin p s, by Lonza (2 mentions)
Roswell park memorial institute (rpmi), by Lonza (2 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Ecacc, by European Collection of Cell Cultures (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Psn 1, by European Collection of Cell Cultures (1 mentions)
A2780cis, by European Collection of Cell Cultures (1 mentions)
Hepg2, by European Collection of Cell Cultures (1 mentions)
8 protocols using skov3
1
Butein Cytotoxicity Assay on A2780 and SKOV3 Cell Lines
2
Ovarian Cancer Cell Line Maintenance
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Ovarian cancer cell lines SKOV3 and A2780 were purchased from the European Collection of Cell Cultures (ECACC®) via Sigma. OVCAR-5 and IGVOA-1 were obtained from Dr W. Cliby (William Cliby, MD, Chair, Division of surgery, The Mayo Clinic, Rochester, Minnesota). SKOV3 was maintained in DMEM/F12 (Invitrogen, Carlsbad, CA, U.S.A.) supplemented with 1% sodium pyruvate (Invitrogen), 0.2% non-essential amino acids (Invitrogen), and 5% FBS in a humidified atmosphere containing 5% CO2 at 37°C. A2780 was cultured in RPMI1640 with 2 mM glutamine and 10% FBS. OVCAR-5 was cultured in DMEM, 10% FBS, 1% PSG, and 0.1 mM non-essential amino acids. IGROV-1 was cultured with DMEM with 10% FBS. For generation of HER or p65 knockdown cell line, SKOV3 transfected with pSIH-H1-p65 plasmid or pSIH-H1-HER2 plasmid was screened using puromycin for 15 days. The survived single clones were picked up and expanded. Expression of HER2 or p65 was examined by Western. The clones with high efficiency of HER2 or p65 knockdown were used for experiments in the present study.
3
Cytotoxicity Profiling of Stilbenes
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Colorectal cancer cells SW480, breast cancer cells MDA-MB-231, and lung cancer cells A549 were purchased from the American Type Culture Collection (Rockville, MD) while ovarian cancer cells SKOV-3 and pancreatic cancer cells PSN-1 were obtained from the European Collection of Cell Cultures (Porton Down, UK). All cancer cell lines were propagated in RPMI-1640 medium (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL).
The half maximal inhibitory concentrations (IC50) for 1 –7 stilbenes were calculated in GraphPad Prism 5 programme.
The half maximal inhibitory concentrations (IC50) for
4
Cell Culture Maintenance: Protocols
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Human cells lines (A2780, SW626, SKOV3, A2780Cis, A2780ADR, A549, HEPG2, OE19, PC3, HCT116) were purchased from the European Collection of Cell Cultures (ECACC) and tested at regular intervals to confirm mycoplasma free status. Cells were grown as adherent monolayers using RPMI-1640 culture medium supplemented with 10% v/v fetal calf serum, 1% v/v penicillin/streptomycin antibiotics, and 1% v/v 2 mM glutamine. Cells were maintained using 25 or 75 cm2 flasks at 37 °C in a humidified atmosphere containing 5% CO2 and passaged at regular intervals using trypsin-EDTA upon reaching 80–90% confluence.
5
Stable Knockdown of SIK3 in Ovarian Cancer Cells
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Two serous-type ovarian cancer cell lines, namely, OVCAR4, purchased from American Type Culture Collection (ATCC; Manassas, VA USA), and SKOV3, purchased from the European Collection of Cell Cultures (ECACC; Salisbury, Scotland), were used. OVCAR4 and SKOV3 cells were cultured in RPMI 1640 and McCoy's 5A medium, respectively, supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml)/streptomycin (100 mg/ml) (Invitrogen, Carlsbad, CA, USA). To generate cells with stable knockdown of SIK3, we transfected OVCAR4 and SKOV3 cells with SIK3-specific small hairpin RNA (strain#01: 5'-CCGGGAAGCATTGGTGCGCTATTTGCTCGAGCAAATAGCGCACCAATGCTTCTTTTTG-3' and strain#61: 5'-CCGGGCCAGGCTTTATCTTATCAAACTCGAGTTTGATAAGATAAAGCCTGGCTTTTTG-3') (National RNAi Core Facility, Academia Sinica, Taiwan). Simultaneously, their corresponding control cells were also established by transfection with the pLKO.1-luciferase vector (Luc) and selection in medium containing 2.5 mg/ml puromycin. After limiting dilution, the expression levels in individual cell clones were confirmed by immunoblotting analyses.
6
Ovarian Cancer Cell Line Culturing and Manipulation
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A2780, SKOV3 and OVCAR-3 cells were purchased from the European Collection of Cell Cultures. Hey cells were donated by Susan Horwitz (Albert Einstein Medical College). SKOV6 and OV2774 were kindly donated by Dr. Kunle Odunsi (Roswell Park Cancer Institute, Buffalo NY). Culture media were selected according to the suggestions of European Collection of Cell Cultures. Glucose-free RPMI (Gibco) medium was used for hypoglycemia experiments. Growth experiments were performed as previously described [Raspaglio 2010]. Silencing of ZEB2 gene expression was obtained by transfection with Transfectin (Bio-Rad) and specific siRNAs (siZEB2), while a non targeting siRNA pool was used as control (siC) (Dharmacon, Lafayette, CO). Transfection with siHuR and siC oligonucleotide duplex were performed as previously described [22 (link)].
7
Cell Culture of Cancer Cell Lines
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The cancer cell lines AsPC-1 and MIA PaCa-2 were purchased from the American Type Culture Collection (UK). The cancer cell lines A2780 and SK-OV-3 were purchased from the European Collection of Cell Cultures (UK). All cells were grown using distributors' instructions. All cells were cultured in either IMDM, McCoy's 5a Medium Modified or RPMI (Lonza, UK) substituted with 10% FBS (15% for McCoy's 5a Medium Modified) (Bio-Sera, UK) and (v/v); 100 units/mL penicillin, 100 µg/mL streptomycin (P/S) (Lonza, UK). All serum was filtered using a 0.2 µM syringe filter prior to addition to media. When not in use all media was stored between 4-6 °C. All cells were incubated at 37 °C in a 5% CO2 atmosphere. Cells were cultured in tissue culture flasks (Sarstedt, UK) and removed via scraping when cells were 70-90% confluent.
8
Cell Line Cultivation and Maintenance
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The cell lines AsPC-1, Caco-2, Colo320, HCT116, JJN3, Lovo, MCF7, MDA-MB-231, MIA PaCa-2, MM.1S and U266B1 were purchased from the American Type Culture Collection (UK). The cell lines A2780, SK-OV-3, T47D and U937 were purchased from European Collection of Cell Cultures (UK). All cell lines were cultured in accordance with distributor's recommendations. Each cell line was cultured using either DMEM, RPMI, IMDM or McCoy's 5a Medium Modified (Lonza, UK) with FBS (Bio-Sera, UK) and (v/v); 100 units/ml penicillin, 100 µg/ml streptomycin (P/S) (Lonza, UK). All serum, P/S and buffers added to media were filtered through a 0.2µm filter before addition. Between use all media was stored at 2-8°C. All cell lines were incubated at 37°C in a 5% CO2 atmosphere except for MDA-MB-231 which was incubated at 37°C in a 0% CO2 atmosphere. All cell lines were cultured in tissue culture flasks (Sarstedt, UK) and removed when cells were either adherent and in the logarithmic growth phase or removed when at a high enough growth density for suspension cell lines.
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