Ewing sarcoma cell lines TC-71 (RRID: CVCL_2213 and SK-N-MC RRID: CVCL_0530) were purchased from DSMZ (Braunschweig, Germany). LAP-35 (RRID: CVCL_A096) was a generous gift from Drs. Katia Scotlandi and Cristina Manara. The absence of mycoplasma contamination was verified every two months by PCR analysis. Cells were maintained in culture in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO—Thermo Fisher Scientific, Waltham, USA, Massachusetts), supplemented with 10% fetal bovine serum, and penicillin and streptomycin (GIBCO) and maintained at 37 °C in humidified 5% CO2 atmosphere. For doxorubicin treatment, Ewing sarcoma cells were treated for the indicated time with either DMSO or the indicated concentrations of doxorubicin (ranging from 0.1 nM to 150 nM).
Tc 71
Manufactured by Leibniz Institute DSMZ
Sourced in Germany, France
The TC-71 is a laboratory equipment used for the high-throughput cultivation and screening of microorganisms. It is designed to automate the process of inoculating, incubating, and analyzing microbial cultures. The core function of the TC-71 is to provide a controlled and standardized environment for the growth and analysis of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Sk n mc, by Leibniz Institute DSMZ (6 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Rd es, by American Type Culture Collection (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Sk n mc, by American Type Culture Collection (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Mhh es1, by Leibniz Institute DSMZ (3 mentions)
Cado es 1, by Leibniz Institute DSMZ (2 mentions)
Rd es, by Leibniz Institute DSMZ (2 mentions)
Sk es 1, by American Type Culture Collection (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Heparin, by Merck Group (1 mentions)
Iscove modified dulbecco media (imdm), by Euroclone (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Lonza (1 mentions)
Ciprofloxacin, by Fresenius (1 mentions)
15 protocols using tc 71
1
Doxorubicin Sensitivity in Ewing Sarcoma
2
Ewing Sarcoma Cell Line Characterization
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Ewing sarcoma cell lines TC-71 (RRID:CVCL_2213) and SK-N-MC (RRID:CVCL_0530) were purchased from DSMZ. Cell were grown in culture in Iscove’s modified Dulbecco’s medium (IMDM, GIBCO), supplemented with 10% fetal bovine serum, penicillin, and streptomycin (Gibco) in a humidified 37 °C incubator with a 5% CO2 atmosphere. Every two months, PCR analysis was performed to evaluate mycoplasma contamination. For the pan-inhibitor screening, Ewing sarcoma cells were treated for the indicated time with either DMSO or the indicated drug (see Table 1 ). Drugs were purchased from Selleckchem. Drug concentration: Afatinib (1 µM), Lapatinib (5 µM), Alisertib (5 µM), Barasertib (100 nM), Tozasertib (100 nM), PD0332991 (1 µM), Belnacasan (10 µM), Navitoclax (1 µM), KU-55933 (10 µM), JNK-IN-8 (1 µM), PF-562271 (1 µM), CPI-455 (10 µM), Etoposide (500 nM–10 mM), and BEZ-235 (1 µM). For UV light irradiation, cells were plated at 50–60% confluence 16 h before UV light irradiation (40 J/m2). Fresh medium was immediately added after the treatment and the cells were harvested after 6 h. Actinomycin D 10 µg/mL was used for the time course experiment.
3
Cultivating and Characterizing Human ES Cell Lines
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The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy). RD-ES cell line (RRID: CVCL_2169, ATCC, LGC Standards S.r.l., Milan, Italy) was maintained in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Media were supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% l -glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl). Human adipose (AD)-MSCs were isolated from healthy donors (n = 2) as previously described [14] (link). After isolation, cells were grown in minimal essential medium with alpha modifications (α-MEM, Gibco) containing up to 5% platelet lysate (PL, Macopharma, Tourcoing, France), 1% L -glutamine, 0.5% ciprofloxacin (Fresenius Kabi Italia S.r.l., Verona, Italy), and 0.2% heparin (Sigma-Aldrich, Saint Louis, Missouri, USA). Cells were incubated and maintained within a controlled atmosphere with 5% CO2 and a temperature of 37 °C. The authentication of TC71, A673, and RD-ES cell lines was recently performed by the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH.
4
Ewing Sarcoma Cell Line Protocol
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EwS cell lines were grown as adherent cultures on collagen-coated flasks in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Thermo Fisher, Dreieich, Germany) and 2 mM L-glutamin (Sigma-Aldrich, Taufkirchen, Germany) and maintained at 37 °C and 5% CO2. TC-32, TTC-466, 5838, A4573 were gifts from the Children’s Hospital Los Angeles, United States. CADO-ES-1 (#ACC 255), RD-ES (#ACC 260), TC-71 (#ACC 516) and NTERA-2 (#ACC 527) were purchased from DSMZ (Braunschweig, Germany). A673 (#CRL-1598) and SK-N-MC (#HTB-10) from ATCC (Manassas, Virginia, USA). Cell lines MS-EwS-6, MS-EwS-15 and MS-EwS-16 were established in our institution, as reported previously31 (link), and WE-68 and VH-64 were gifts from the Institute of Experimental Orthopedics of our institution. Control cell lines SUP-B15 (B lineage leukemia) (#ACC 389) and HT-1080 (fibrosarcoma) (#ACC 315) were purchased from DSMZ and were cultivated in RMPI 1640 medium supplemented with 10% heat-inactivated FBS and 2 mM l -glutamin. Short tandem repeat (STR) profiling was used to confirm the identity of all cell lines (Supplementary Table 1 ), and all cell lines were regularly checked by PCR to exclude mycobacterial contamination.
5
Cell Culture Protocols for ELISA
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The THP-1 cell line is a monocytic leukemia cell line and was purchased from the German Collection of Microorganism and Cell Cultures (DSMZ, Braunschweig, Germany). The cell line was cultivated as recommended in RPMI 1640 with supplements and subcultivated every three to four days. The human myeloid leukemia cell line HL-60 was obtained from the DSMZ (Braunschweig, Germany). The cultivation was carried out in RPMI 1640 with supplements according to the specifications. For the ELISA both cell lines were plated at 2.4 x 105 cells/well into 24-well plates, final volume 400 μL. The cells were incubated for 24 h at 37°C with 5% CO2.
The 143B cell line is a human osteosarcoma cell line and was purchased from the American Tissue Culture Collection (ATCC, Wesel, Germany). The cells were maintained as recommended in DMEM with supplements. The Ewing’s sarcoma cell line TC-71 was obtained from DSMZ (Braunschweig, Germany) and was cultivated as recommended every two to three days in IMDM with supplements. For the ELISA the adherent cell lines were seeded at 1.2 x 105 cells/well into 24-well plates and incubated as well for 24 h at 37°C with 5% CO2.
The 143B cell line is a human osteosarcoma cell line and was purchased from the American Tissue Culture Collection (ATCC, Wesel, Germany). The cells were maintained as recommended in DMEM with supplements. The Ewing’s sarcoma cell line TC-71 was obtained from DSMZ (Braunschweig, Germany) and was cultivated as recommended every two to three days in IMDM with supplements. For the ELISA the adherent cell lines were seeded at 1.2 x 105 cells/well into 24-well plates and incubated as well for 24 h at 37°C with 5% CO2.
6
Sourcing Human Cancer Cell Lines
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Human neuroblastoma cells lines Kelly and LAN1 and the human Ewing's sarcoma cell lines A673 and Rh1 were originally obtained from the ATCC. The Ewing's sarcoma cell lines TC-71 and Cado were from DSMZ (Braunschweig, Germany). VH-64 and WE-68 cells were gifts from Frans van Valen's laboratory at the Institute of Experimental Orthopedics of University of Muenster, Germany. A-4573 and TC-32 were from the cell line bank at Children's Hospital Los Angeles. MS-PES4 and DC-ES6 were established by our group as described previously.14 (link)
7
Characterization of Ewing's Sarcoma Cell Lines
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The following EwS cell lines were used: VH-64 and WE-68 were a gift from Frans van Valen´s laboratory at the Institute of Experimental Orthopedics of University of Muenster, Germany). A4573, 5838, TTC-466 and TC-32 were kindly provided by the Children’s Hospital Los Angeles. RD-ES, TC-71, SK-ES1 and CADO-ES-1 were obtained from DSMZ (Braunschweig, Germany). MS-PES-1, MS-PES-4, MS-PES-6, and DC-ES-6 were established in our lab from biopsy material obtained upon metastatic relapse. K562 is a human leukemia cell line, and JEG-3 is a human placental choriocarcinoma cell line (both from DSMZ). All tumor cells were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal calf serum (FCS; Thermo Fisher, Bonn, Germany) and 2 mM L-glutamine (PAA, Cölbe, Germany), and maintained at 37°C and 5% CO2. EwS cell lines TC-32, TC-71, and 5838 were cultured in uncoated flasks, and all other EwS cell lines were cultured in collagen-coated 25 cm2 tissue culture flasks. The identity of the cell lines was confirmed by short tandem repeat (STR) profiling. For some experiments, EwS cell cultures with 30-50% confluence were stimulated with medium containing 500 U/ml IFN-γ for 48 hours.
8
Transduction of cancer cell lines
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The neuroblastoma cell line JF was kindly provided by Malcolm K. Brenner, Houston, USA. The Ewing’s sarcoma cell line TC-71 was obtained from DSMZ (Braunschweig, Germany). The prostate cancer cell line PC3 as well as the CHO cell line were purchased from American Type Culture Collection. For the bioluminescence based killing assay and the in vivo analysis JF and TC-71 cells were transduced to express the gene encoding firefly luciferase (JF Luc, TC-71 luc). Transduction was performed using a lentiviral packaging system as described previously [39 (link)]. PC3 cells expressing firefly luciferase were described previously [36 (link)]. All these cell lines were cultured in RPMI 1640 medium completed with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin, 2 mM N-acetyl-L-alanyl-L-glutamine, 1% non-essential amino acids and 1 mM sodium pyruvate (Biochrom). Human Embryonic Kidney cells HEK293T (ATCC CRL-11268) used for production of the lentiviral particles were cultured in DMEM medium supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 1% non-essential amino acids (Biochrom). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
9
Characterization of Ewing Sarcoma Cell Lines
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EwS lines (MHH-ES1, SKNMC, and TC-71), were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). A673 was purchased from ATCC (LGC Standards, Teddington, UK). EW7 cells were kindly provided by O. Delattre (Institut Curie, Paris, France). Cells were maintained in a humidified incubator at 37 °C in 5% CO2 atmosphere in RPMI 1640 (Life Technologies, Darmstadt, Germany) containing 10% heat-inactivated fetal bovine serum (Life Technologies) and antibiotics (Life Technologies). Cell lines were routinely checked for purity (e.g., EWS-FLI1 translocation product, surface antigen, or HLA-phenotype) and mycoplasma contamination as well as for identity by DNA profiling using 8 different and highly polymorphic short tandem repeat (STR) loci (DSMZ, Braunschweig, Germany).
10
Ewing Sarcoma Cell Line Provenance
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