SK-N-AS, SK-N-FI, SK-N-BE(2)-C, SH-EP, SH-SY5Y, IMR-32, H460, A549, DU145, HCT116, HT29, PC3, SKOV3, CHP-134, Kelly, MDA-MB-231, and HEK-293T cells were purchased from ATCC by the Children’s Cancer Institute Australia Tumour Bank, and maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Australia) with 10% fetal calf serum. The human lung fibroblast cells MRC-5 and WI-38 (ATCC) were cultured in Alpha-MEM media with 10% FCS. Non-MYCN-amplified human neuroblastoma cell lines SH-EP MYCN-3 and SHEP-tet21N, which are genetically modified to overexpress or repress human MYCN cDNA when exposed to doxycycline, were generously supplied by Professor Jason Shohet (Texas Children’s Cancer Center, Houston). The shMYCN SK-N-BE(2)-C cell line are genetically modified to repress human MYCN expression by MYCN shRNA when exposed to doxycycline, and SK-N-BE(2)-C cell line was used as the parental cell line. The shMYCN SK-N-BE(2)-C cell line was derived by GenScript, Piscataway NJ. All cell lines used were authenticated by CellBank Australia (Westmead, NSW), to be free from mycoplasma, and were cultured at 37 °C with 5% CO2 in a humidifier incubator.
Sh ep
Manufactured by American Type Culture Collection
Sourced in Australia, United Kingdom, Switzerland, United States
The SH-EP is a piece of laboratory equipment designed for the efficient storage and preservation of cell cultures. It provides a controlled environment to maintain the viability and integrity of cell lines during storage and transport.
Automatically generated - may contain errors
Lab products found in correlation
Sk n as, by American Type Culture Collection (7 mentions)
Sh sy5y, by American Type Culture Collection (5 mentions)
Imr 32, by American Type Culture Collection (4 mentions)
Sk n sh, by American Type Culture Collection (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Sk n be 2 c, by American Type Culture Collection (3 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Sk n be 2, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Be 2 c, by American Type Culture Collection (2 mentions)
Penicillin, by Euroclone (2 mentions)
Streptomycin, by Euroclone (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Sk n fi, by American Type Culture Collection (1 mentions)
Wi 38, by American Type Culture Collection (1 mentions)
Chp134, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
Mrc 5, by American Type Culture Collection (1 mentions)
11 protocols using sh ep
1
Cell Lines for Neuroblastoma Research
2
Comprehensive Analysis of 12q-Amplified Neuroblastoma
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The Swedish samples (n = 436) were screened using Affymetrix SNP microarrays (Thermo Fisher Scientific, Waltham, MA) as described earlier7 (link),44 (link). In addition, 12 NB cell lines were also screened using SNP microarray analysis: IMR32, KELLY, NB69, SH-SY5Y, SK-N-AS, SK-N-BE(2), SK-N-DZ, SK-N-FI, SK-N-SH (ECACC, HPA Culture Collections, Salisbury, UK), SH-EP (ATCC, Manassas, VA), as well as the two 12q-amplified cell lines LS and NGP (kindly provided by Prof. Manfred Schwab, DKFZ Heidelberg, Germany). There were nine patients with tumors displaying high-grade 12q-amplification in the Swedish cohort. In addition to these samples, a selection of patients from Norway (n = 1), Spain (n = 3), Austria (n = 2), and Poland (n = 2) with tumors sharing a similar 12q-amplification pattern were also included (Supplementary Fig. 1 ). In total, 19 cases with 12q-amplification were included in the study (tumor material from 17 patients and two cell lines). For primary data analysis, GDAS software (Thermo Fisher Scientific) was used, while genomic profiles and amplicon boundaries were determined using either the Chromosome Analysis Suite (ChAS 3.3; Thermo Fisher Scientific) or the Copy Number Analyzer for Affymetrix GeneChip Mapping arrays (CNAG 3.0; Genome Laboratory, Tokyo, Japan; www.genome.umin.jp ).
3
Cell Line Cultivation for Neuroblastoma Research
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The NB cell line SH-EP was obtained from N. Gross, Lausanne, Switzerland [25 (link)] and the NB cell lines SK-N-SH and IMR32 were purchased from ATCC (Rockville, MD, USA). For all cell culture experiments with these cells, RPMI1640 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (Sigma-Aldrich, Vienna, Austria), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (Lonza, Basel, Switzerland) was used. PhoenixTM [26 (link)] and HEK293T packaging cells were cultivated in DMEM medium (Lonza, Basel, Switzerland). Using the VenorRGeM-mycoplasma detection kit (Minerva Biolabs, Berlin, Germany), all cells were routinely tested for mycoplasma contamination. All reagents were purchased at Sigma-Aldrich (Vienna, Austria) unless stated otherwise.
4
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].
5
Cell Line Handling and Authentication
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NIH3T3, HEK293T, BE2C, and SHEP were purchased from the American Type Culture Collection. Kelly cells were purchased from Sigma-Aldrich. NB-19, SK-N-FI, and CHLA-90 cells were a gift from Y. DeClerck (Children's Hospital Los Angeles, Los Angeles, USA). NB-SD, NMB, NB69, and SK-N-SH cells were obtained from the Children’s Hospital of Philadelphia (CHOP) cell line bank. Tet21N cells were a gift from M. Schwab (German Cancer Research Center, Heidelberg, Germany). NIH3T3 cells stably expressing the empty vector Murine Stem Cell Virus (MSCV), oncogenic EN (ETV6-NTRK3), or mutant KRasV12 were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) with 10% bovine serum (calf serum, Gibco). All cell lines have been authenticated by short tandem repeat (STR) profiling using the AmpFLST Identifiler PCR Amplification Kit (Applied Biosystems) and were tested for mycoplasma on a regular basis using the LookOut Mycoplasma Detection Kit (Sigma-Aldrich). HEK293T cells were cultured in DMEM with 10% fetal bovine serum (FBS; Gibco). All other cell lines were cultured in RPMI 1640 supplemented with 10% FBS. All media were supplemented with 1% antibiotic-antimycotic (Gibco), and all cells were cultured at 37°C with 5% CO2.
6
Characterization of Human Neuroblastoma Cell Lines
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Human NB cell lines were obtained as follow: GICAN and ACN from Interlab Cell Line Collection, Banca Biologica and Cell Factory (www.iclc.it ), SK-N-AS, SH-SY5Y, SH-EP, SK-N-SH, SK-N-BE(2)c, IMR-32 from the American Type Culture Collection (ATCC), LA-N-1 from Creative Bioarray, LA-N-5 from the Leibniz-Institut DMSZ, SMS-KCNR from Children's Oncology Group Cell Culture, while the Tet-21/N cell line was kindly provided by Dr M. Schwab (University of Heidelberg, Heidelberg, Germany). All NB cell lines were characterized by (i) HLA class I typing by PCR-SSP sets (Genovision) according to the manufacturer's instructions, and (ii) array CGH (see below). The human erythro-leukemia cell line K562 was purchased from ATCC and used as control target for NK cell functional assays. Cells were grown in RPMI 1640 medium supplemented with 10% FBS (Thermo Fisher Scientific), 2 mM glutamine, 100 mg/mL penicillin and 50 mg/mL streptomycin (Euro Clone S.p.a.). Doxycycline (Sigma Aldrich) was used at 10 ng/mL. Lipofectamine 2000 was used, according to manufacturer's instructions (Invitrogen), to transfect SK-N-SH cells with pIRVneoSV empty vector or pIRVneoSV-MYCN, both kindly provided by G. Giannini (“La Sapienza” University of Rome, Italy).
7
Characterization of NB Cell Lines
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Human NB cell lines were obtained as follows: SK-N-AS, SH-SY5Y, SH-EP, SK-N-SH, SK-N-BE(2)c, and IMR-32 from the American Type Culture Collection (ATCC) and LA-N-5 from the Leibniz-Institut DSMZ. All NB cell lines were characterized by (i) HLA class I typing by PCR-SSP sets (Genovision) according to the instructions of the manufacturer and (ii) array comparative genomic hybridization (a-CGH) and single-nucleotide polymorphism (SNP) array analyses (see below). The human non-small-cell lung cancer cell line A549 was purchased from Sigma-Aldrich. The human erythroleukemia cell line K562 was purchased from ATCC and used as a control target for NK cell functional assays. Cells were grown in RPMI 1640 medium supplemented with 10% FBS (Thermo Fisher Scientific), 2 mM glutamine, 100 mg/ml penicillin, and 50 mg/ml streptomycin (EuroClone S.p.A.). Cisplatin (Accord Healthcare Limited), etoposide (Teva Italia), irinotecan (Campo, Pfizer), and topotecan (GlaxoSmithKline) were kindly provided by the pharmacy of our institution.
8
Cell Line Authentication and Validation
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Human HEK293T, SK-N-BE, SK-N-AS, and SH-EP cell lines were obtained from the American Type Culture Collection (respectively ATCC #CRL-3216, #CRL-2268, #CRL-2137, #CRL-2269). The cell lines used for all experiments were re-authenticated and tested as mycoplasma-free and grown in commercial media, as reported in the supplemental information . Early passage cells were used, and the cumulative culture length was less than 3 months after resuscitation.
9
Neuroblastoma Cell Lines and NK Cell ADCC
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The human neuroblastoma cell lines BE(2)-C, SK-N-AS, SH-SY5Y, SHEP, SK-N-DZ, and LAN-1 were purchased from the American Type Culture Collection (Manassas, VA). Cells were maintained in Rockwell Park Memorial Institute (RPMI) culture medium 1640 with 10% fetal bovine serum at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. Our laboratory previously established the stable luciferase-expressing neuroblastoma cell lines, BE(2)-C/Luc and SK-N-AS/Luc, which were selected and cultured in zeocin (50 μg/ml) contained medium. BE(2)-C/shCON, which functioned as a control and GRP-R shRNA knock-down cells (shGRP-R) were both established previously [26 (link)]. Natural killer (NK) cells were purchased from Lonza, cultured in Lymphocyte Growth Media-3 (LGM-3, Lonza), and stimulated by IL-2 (100 U/ml) supplemented in the media. NK cell purity was confirmed with flow cytometry by staining for CD3-CD56+. These activated NK cells were then used for GRP-R mAb-mediated antibody-dependent cellular cytotoxicity (ADCC) assays.
10
MYCN-Dependent Cell Line Cultivation
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NB cell lines with different MYCN status (SH-EP: MYCN non-amplified; LAN-5: MYCN amplified; NLF: MYCN amplified) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in DMEM GlutaMAX™-l or RPMI medium (Life Technologies, Inc., Darmstadt, Germany), supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1 mM sodium pyruvate and 10 mM HEPES (all from Life Technologies, Inc.). LAN-5 cells were grown in flasks coated with collagen (BD Biosciences, San Jose, CA, USA). VCR, VBL and VNR were purchased from Sigma (Deisenhofen, Germany); RO-3306 from Merck (Darmstadt, Germany); zVAD.fmk from Bachem (Heidelberg, Germany). MCL-1 plasmids (pCMV-Tag 3B, MCL-1 ‘4A’) were kindly provided by Genentech, Inc. (South San Francisco, CA, USA). Chemicals were purchased from Sigma unless otherwise indicated.
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