MycCaP, PC3, LNCaP, PC12, MV411, SK-N-BE (2) and 293T cells lines were purchased from ATCC and P493–6 B cells were kindly provided by Professor Chi Van Dang from the University of Pennsylvania. LLC1 cells were from Professor Bin Zhang (Northwestern University). TGR-1 and HO15.19 Rat-1 cells were gift from Professor John Sedivy in Brown University. Cells were verified to be mycoplasma-free (Lonza) at multiple times throughout the study. MycCaP, PC3, LNCaP and P493–6 B cells were cultured in RPMI1640; LLC1 and 293T cells in DMEM (Gibco); SK-N-BE(2) cells in F12 (ATCC); MV411 cells in IMDM (ATCC), all supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco), 1% Penicillin-Streptomycin (10,000U/ml, Life Technologies). PC12 cells were grown in F-12K Medium (ATCC) with 2% heat inactivated FBS, 12.5% of horse serum (Thermo), and Rat-1 cells were cultured in DMEM with 10% calf serum. All cell culture was performed in a 37°C 5% CO2 incubator.
Sk n be 2
Manufactured by American Type Culture Collection
Sourced in United States
SK-N-BE(2) is a human neuroblastoma cell line derived from a bone marrow biopsy. It is a commonly used model for the study of neuroblastoma, a type of cancer that originates in the nerve tissue.
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Lab products found in correlation
Sh sy5y, by American Type Culture Collection (24 mentions)
Sk n as, by American Type Culture Collection (21 mentions)
Sk n sh, by American Type Culture Collection (19 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (17 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (12 mentions)
Imr 32, by American Type Culture Collection (12 mentions)
Sk n dz, by American Type Culture Collection (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (3 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Co2 incubator, by Thermo Fisher Scientific (3 mentions)
Ham s f 12, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Merck Group (2 mentions)
61 protocols using sk n be 2
1
Cell Culture Protocols in Cancer Research
2
Maintenance of Human Neuroblastoma Cell Lines
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The human neuroblastoma cell lines SK-N-AS (CRL-2137) and SK-N-BE(2) (CRL-2271) were obtained from American Type Culture Collection (ATCC, Manassas, VA). SH-EP and WAC(2) human neuroblastoma cell lines were a kind gift from M. Schwab (Deutsches Krebsforschungszentrum, Heidelberg, Germany) and have been described in detail [13] (link). All cell lines were maintained under standard conditions at 37°C and 5% CO2. SK-N-AS cells were maintained in Dulbecco's modified Eagle's medium (DMEM, 30-2601, ATCC) containing 10% fetal bovine serum (Hyclone, Suwanee, GA), 4 mM L-glutamine (Thermo Fisher Scientific Inc., Waltham, MA), 1 μM nonessential amino acids and 1 μg/ml penicillin/streptomycin (Gibco, Carlsbad, CA). SK-N-BE(2) cells were maintained in a 1:1 mixture of minimum Eagle's medium and Ham's F-12 medium (30-2004, ATCC) with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine (Thermo Fisher Scientific), 1 μM nonessential amino acids and 1 μg/ml penicillin/streptomycin (Gibco). SH-EP and WAC2 cell lines were maintained in RPMI 1640 medium (30-2001, ATCC) with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine (Thermo Fisher Scientific) and 1 μg/ml penicillin/streptomycin (Gibco). All four cell lines were verified within the last 12 months using short tandem repeat analysis [Heflin Center for Genomic Sciences, University of Alabama, Birmingham (UAB), Birmingham, AL].
3
Characterization of NB Cell Lines
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Two NB cell lines, namely SH-SY5Y (ATCC CRL-2266) and SK-N-BE(2) (ATCC CRL-2271), were selected for the xenograft studies. SH-SY5Y is a TP53 wild type, non-MYCN-amplified cell line with no chromosome 1p loss of heterozygosity. The SK-N-BE(2) cell line is amplified for MYCN, carries a TP53 mutation (p.C135F), and is heterozygous for loss of chromosome 1p [70 (link)]. Expression of MYCN protein in these cell lines was reported earlier [42 (link)]. NB cells were cultured under standard conditions, as described previously [41 (link)].
4
Neuroblastoma cell lines culture
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Neuroblastoma cell lines SK-N-AS (non-MYCN-amplified, high GLI1 expression) and SK-N-BE(2) (MYCN-amplified, low GLI1-expression) [19 (link), 23 (link), 24 (link)], obtained from ATCC (Manassas, VA), were cultured in RPMI-1640 with 10% fetal calf serum and 100 IU/ml penicillin/streptomycin and maintained in a 5% CO2 humidified incubator. RPMI-1640, penicillin/streptomycin, and trypsin were purchased from Invitrogen. Recombinant tumor necrosis factor alpha (hTNF-α) was obtained from Roche Applied Sciences.
5
Neuroblastoma Cell Lines Cultured and Treated
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Human neuroblastoma cell lines SK-N-BE2, SH-SY5Y, and IMR-32 were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM), MEM-F12, or MEM (contained 10 mM NEAA) supplemented with 10% (v/v) fetal bovine serum (Biological industries). All cells were maintained at 37°C in a humidified incubator (Thermo Scientific) with 5% CO2. Critical chemicals used in this study were shown as follows: Cisplatin (MedChemExpress, HY-17394), U-104 (MedChemExpress, HY-13513), and Actinomycin D (MedChemExpress, HY-17559). The Cisplatin treatment concentrations of SK-N-BE(2), SH-SY5Y, and IMR-32 cells were based on their IC50s of Cisplatin. The dosing time of Cisplatin treatment was 24 h.
6
Neuroblastoma Clinical and Cell Line Study
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Patients diagnosed with neuroblastoma in Xin Hua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, in the period of Sep. 2016 to July 2019, were enrolled to collect clinical information and tumor samples for further investigation. All patients were treated according to the Chinese Children Cancer Group-NB-2014 (CCCG-NB-2014) protocol [13 ]. This study was approved by the Ethics Committee of Xin Hua Hospital affiliated with Shanghai Jiao Tong University School of Medicine. The cell lines SK-N-SH, IMR-32, SH-SY5Y, and 293T were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). SK-N-BE(2) and SK-N-AS were purchased from ATCC (Manassas, USA). SK-N-SH, IMR-32, SK-N-BE(2), and 293T were all cultured in DMEM supplemented with 10% fetal bovine serum(FBS) and 1% penicillin-streptomycin solution. SH-SY5Y and SK-N-AS cell lines were cultured in a 1:1 mixture of MEM and F12 Medium with 1% Gluta-max, 1% Sodium pyruvate, 1% NEAA, 1% penicillin-streptomycin solution, and 10% FBS. The mediums and FBS were all purchased from Gibco, USA. All cells grew in a humidified incubator with 5% CO2 at 37°C.
7
Neuroblastoma Cell Culture and Metformin Treatment
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Human neuroblastoma SK-N-BE(2) and SH-SY5Y cells (ATCC, Manassas, VA) were maintained in complete culture medium (Dulbecco's Modified Eagle's Medium, DMEM + 10% fetal bovine serum, FBS; Atlanta Biologicals, Lawrenceville, GA) at 37°C in a humidified incubator with 5% CO2. Stock solution of metformin (MP Biomedicals, Solon, OH) was freshly prepared in sterile triple distilled water before each experiment. Cells treated with equal volume of vehicle were used as control.
8
Bovine MEV Treatment on Cell Lines
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The NBL cell line, SK-N-BE2 (ATCC, Virginia, USA) and the colon cancer cell line C26 (gifted by Prof. Hoogenraad) were cultured in 150 cm2 tissue culture flask (BD Falcon™) in Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) (GIBCO, Life Technologies) medium, respectively. The medium was supplemented with 10% (v/v) fetal calf serum (FCS) (GIBCO, Life Technologies) and 100 units/mL of penicillin-streptomycin (GIBCO, Life Technologies). The cells were incubated at 37 °C with 5% CO2 and were treated with 100 µg/mL of bovine MEVs. The cells were lysed using SDS lysis buffer (2% (w/v) SDS, 125 mM Tris-HCl pH 7.4, 12.5% (v/v) glycerol, and 0.02% (w/v) bromophenol blue) at 24, 48 and 72 h.
9
Neuroblastoma Cell Line Experiments
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The neuroblastoma cell line SH-SY5Y was purchased from DSMZ (Braunschweig, Germany), while SK-N-BE(2) and IMR-32 cells were obtained from ATCC (Manassas, VA). The cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% antibiotics, and glutamine (Gibco). The cells were then exposed to PON (Sigma-Aldrich), CQ (Sigma-Aldrich), or a combination of PON and CQ (COMBO) for the indicated times and at the specified doses. The genetic background of the cell lines is summarized in Supplementary Table S1 . In vivo studies were done with the less toxic analog hydroxychloroquine (HCQ; Sigma-Aldrich) [27 (link)]. Torin 1 (Sigma-Aldrich) was dissolved in DMSO before use, and the cell cultures were regularly tested for the presence of mycoplasmas by PCR. Human cell line authentication was done at BMR Genomics S.r.l. (Padova, Italy).
10
Xenograft Studies of Neuroblastoma Cell Lines
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The cell lines SH-SY5Y (ATCC CRL-2266) and SK-N-BE(2) (ATCC CRL-2271) were chosen for the xenograft studies. SH-SY5Y is a TP53 wild-type, non-NMYC-amplified cell line that shows no chromosome 1p loss of heterozygosity and is sensitive to chemotherapy. The SK-N-BE(2) cell line shows high resistance to a wide range of chemotherapeutic agents and is characterized by NMYC amplification, TP53 mutation (p.C135F) and chromosome 1p loss of heterozygosity [48 (link)]. Cells were grown in a 1:1 mixture of Eagle's Minimum Essential Medium and Ham’s F12 (Sigma), supplemented with 10% fetal bovine serum (PAA), Glutamax (Gibco), non-essential amino acids (Sigma) and penicillin/streptomycin/amphotericin (Sigma).
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