In this experiment, the human liposarcoma cell line SW872 (CL-0685B) and the human fibrosarcoma cell line HT1080 (CL-0117) were purchased from Procell Biotech (Wuhan, China), and passed STR identification and mycoplasma contamination testing. In this study, SW872 and HT1080 were cultured in DMEM (Gibco), which was supplemented with 10% FBS (Gibco) and 1% streptomycin/penicillin (Procell Biotech). To overexpress TNFSF14, TNFSF14 were cloned into a pcDNA3.1 vector, then transiently transfected into SW872 and HT1080 cells for 24 h. Lipofectamine 3000 (Invitrogen, USA) was used for transfections according to the protocol.
Ht1080
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
The HT1080 is a laboratory equipment designed for cell culture. It provides a controlled environment for the growth and maintenance of cells. The HT1080 features temperature, humidity, and gas regulation capabilities to support optimal cell culture conditions.
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Lab products found in correlation
Ht1080, by American Type Culture Collection (22 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Mcf 7, by American Type Culture Collection (5 mentions)
Dmem high glucose media, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Sh sy5y, by American Type Culture Collection (4 mentions)
Mcf 7, by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
H1299, by American Type Culture Collection (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Horse serum, by Thermo Fisher Scientific (3 mentions)
Hepg2, by Thermo Fisher Scientific (3 mentions)
Mccoy s 5a media, by Thermo Fisher Scientific (3 mentions)
Calu 1, by American Type Culture Collection (3 mentions)
Penicillin streptomycin pen strep, by Thermo Fisher Scientific (3 mentions)
Medium 199, by Merck Group (3 mentions)
35 protocols using ht1080
1
Overexpression of TNFSF14 in Liposarcoma and Fibrosarcoma
2
Culturing Human Cell Lines for Leukemia Research
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The human bone marrow stromal cell line, HS5, and the human acute myeloid leukemia cell line, U93779 (link), were obtained from the American Type Culture Collection (Manassas, VA, USA). 293FT and HT1080 cells were acquired in 2012 from Invitrogen (Carlsbad, CA, USA). Cells were cultured in appropriated medium, according to the manufacturer’s instruction, containing 10% fetal bovine serum (FBS) and glutamine with penicillin/streptomycin and amphotericin B, and maintained at 37 °C, 5% CO2. Recombinant human TNF-α and IFN-γ were purchased from PeproTech (Rocky Hill, NJ, USA). Cytarabine (AraC) was obtained from Intas Pharmaceuticals (Ahmedabad, India) and prepared as a 10 mM stock solution.
3
Cell Culture Conditions for Cancer and Normal Cell Lines
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Human colorectal adenocarcinoma (SW480), colorectal carcinoma (HCT116), lung adenocarcinoma (A549), and fibrosarcoma (HT1080), and normal bronchial epithelial (BEAS-2B) cells were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Human neuroblastoma (SH-SY5Y) and prostate carcinoma (PC3) cells were maintained in MEM (Invitrogen) supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. SW480, HCT116, A549, HT1080, SH-SY5Y, PC3, and BEAS-2B were purchased from American Type Culture Collection. Human microvascular endothelial cells (HMEC-1s) were cultured in Endothelial Growth Medium-2 (EGM-2; Lonza, Walkersville, MD, USA) [5] (link). Human umbilical vein endothelial cells (HUVECs) were isolated from freshly delivered umbilical cords and maintained as described previously [39] (link). HUVECs were cultured in medium 199 (Invitrogen) supplemented with 20% FBS, 3 ng/mL FGF2 (R&D Systems, Minneapolis, MN, USA), 5 U/mL heparin (Sigma-Aldrich, St Louis, MO, USA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. All cell lines were mycoplasma-free confirmed using e-MyCo Mycoplasma PCR Detection Kit (iNtRON Biotechnology. Inc., Seoul, Korea).
4
Establishing Stable Cell Lines for Protein Expression
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HEK293T, HT-1080 and KYSE-170 cells were purchased from the American Type Culture Collection (ATCC). HEK293T and HT-1080 cells were cultured in DMEM (Invitrogen) supplemented with 5% FBS (Gibco), 100 unit/mL penicillin, and 100 mg/mL streptomycin (Gibco). KYSE-170 cells were cultured in RPMI 1640 medium (Gibco) with 10% FBS, 100 unit/mL penicillin, and 100 mg/mL streptomycin.
Cell transfection was carried out by Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen).
Cells stably expressing the indicated proteins were established by standard retroviral infection, and selected in 2 mg/mL puromycin (Ameresco) or 50 mg/mL hygromycin B (Ameresco) for 7 days. The mutant IDH1 allele knocked out HT-1080(IDH1+/−) cells were generated previously by TALEN technology.
Cell transfection was carried out by Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen).
Cells stably expressing the indicated proteins were established by standard retroviral infection, and selected in 2 mg/mL puromycin (Ameresco) or 50 mg/mL hygromycin B (Ameresco) for 7 days. The mutant IDH1 allele knocked out HT-1080(IDH1+/−) cells were generated previously by TALEN technology.
5
Characterization of ALT+ Cell Lines
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W-V is an SV40 transformed, ALT+ cell line derived from normal fibroblasts from a 45-year-old male with Werner’s syndrome (WRN−/−). W-V was a gift from Roger R. Reddel. The WI38VA13/2RA (VA13/2RA) ALT+ cell line, derived from normal female lung fibroblasts transformed by SV40 large T antigen, was obtained from the European Collection of Authenticated Cell Cultures (ECACC). SUSM-1 ALT+ cell line, derived from male liver fibroblasts immortalized by chemical (4NQO) transformation was a gift from Olivia M. Pereira-Smith. SAOS-2 and U2OS are both female osteosarcoma derived ALT+ cell lines obtained from Paolo Salomoni. HT1080, a telomerase-positive cell line derived from a male fibrosarcoma was obtained from ECACC.
W-V, U2OS and HT1080 were grown in DMEM medium (Gibco, UK) with 10% Fetal Bovine Serum (Gibco, UK); SAOS2 in RPMI 1640 (Gibco, UK) with 10% FBS and VA13/2RA in MEM medium (Gibco, UK) with 10% FBS and 1X Non-Essential Amino Acids (Gibco, UK). The cells were culture at 37 °C in 5% CO2 and ambient O2 and subcultured by standard methods. Dead cells were assessed using Trypan Blue stain (final concentration 0.2%, 3mins).
W-V, U2OS and HT1080 were grown in DMEM medium (Gibco, UK) with 10% Fetal Bovine Serum (Gibco, UK); SAOS2 in RPMI 1640 (Gibco, UK) with 10% FBS and VA13/2RA in MEM medium (Gibco, UK) with 10% FBS and 1X Non-Essential Amino Acids (Gibco, UK). The cells were culture at 37 °C in 5% CO2 and ambient O2 and subcultured by standard methods. Dead cells were assessed using Trypan Blue stain (final concentration 0.2%, 3mins).
6
Comprehensive Cell Line Culture Protocols
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Human cancer cell lines HL-60, K562, Jurkat, HeLa, A549, MCF-7, HepG2, A431, and MIA PaCa-2, along with human normal cell lines WI-38 and 1C3D3, were obtained from the RIKEN Cell Bank (RIKEN BRC, Tsukuba, Japan). Human normal cell line YS-1 was obtained from the JCRB Cell Bank (Osaka, Japan). Human cancer cell lines U937, PC-3, DLD-1, Hep3B, WM266-4, SK-MEL-28, HT-1080, and BxPC-3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). HL-60, K562, Jurkat, U937, MCF-7, DLD-1, and BxPC-3 cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (Sigma-Aldrich), 50 units/mL penicillin G (Gibco), and 50 μg/mL streptomycin (Gibco). HeLa, A549, PC-3, HepG2, Hep3B, WM266-4, SK-MEL-28, HT-1080, A431, MIA PaCa-2, and WI-38 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal calf serum, 50 units/mL penicillin G, and 50 μg/mL streptomycin. YS-1 cells were cultured in DMEM containing 5% fetal calf serum, 50 units/mL penicillin G, and 50 μg/mL streptomycin. 1C3D3 cells were cultured in DMEM containing 5% fetal calf serum, 10% newborn bovine serum (SAFC Biosciences, Lenexa, KS, USA), 2.5% horse serum (Gibco), 50 units/mL penicillin G, and 50 μg/mL streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO2.
7
Culturing Three STS Cell Lines
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Three human STS cell lines (RD ATCC® Number: HTB-166™, SW982 ATCC® Number: HTB-93™ and HT1080 ATCC® Number: CCL-121™ ) were purchased from American Type Culture Collection. RD and HT1080 cells were cultured in RPMI-1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) at 37 °C with a humidified 5% CO2 atmosphere. SW982 cells were cultured in Leibovitz’s L-15 medium (Gibco, CA, USA) containing 10% FBS.
8
Culturing MG63 and HT1080 Cell Lines
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MG63 and HT1080 cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). MG63 was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco™, Thermo). HT1080 was cultured in Minimum Essential Medium (MEM) with Non-Essential Amino Acids (NEAA) (Gibco™, Thermo). The entire medium contained 10% fetal bovine serum (FBS, Gibco™, Thermo), 100 U/ml of penicillin (Sigma) and 100 μg/ml of streptomycin (Sigma). Cells were placed in a CO2 incubator (Series 8000 Water-Jacketed CO2 Incubators, Thermo) maintained at 37°C, 5% CO2, and saturates humidity. At 90% confluence, cells were digested with 0.25% trypsin-EDTA (Gibco™, Thermo) for subculturing.
9
Endoglin Expression in Cell Lines
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The human fibrosarcoma cell line, HT-1080 which overexpresses endogenous human endoglin was purchased from Cell Lines Services (CLS, Heidelberg, Germany). MDA-MB231, a human breast carcinoma cell line with low levels of endogenous human endoglin expression was also purchased from Cell Lines Services. The murine melanoma cell line B16F10 which expresses low levels of endogenous murine endoglin, was stably transfected with a vector DNA bearing murine endoglin gene in previous work [39 (link)] to get the high murine endoglin expressing cell line, B16F10mCD105 used here. The MDA-MB231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) and 1% Hepes (Gibco®, Paisley, Scotland), whereas the HT-1080 cell line, its stable FAP expressing counterpart HT1080-hFAP, and the B16F10mCD105 cells were cultured in RPMI medium supplemented with 5% (v/v) FCS (Gibco®). Standard culture conditions (37 °C, 5% CO2 and 95% humidified atmosphere) were applied for all the cell lines.
10
Lung and Colon Cancer Cell Lines
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H1299 human non-small cell lung cancer, HT1080 human fibrosarcoma, HCT116 human colon, and HeLa human epithelial cervix carcinoma cell lines were purchased from American Type Culture Collection. HeLa and HCT116 were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Euroclone, ECM0728L), H1299 and HT1080 in Roswell Park Memorial Institute medium (RPMI 1640; Gibco, 21875–034), both supplemented with 10% fetal bovine serum. The indicated cell lines were grown in a CO2 humidified incubator at 37 °C. HT1080 and H1299 cells were stably transfected with EGFP-LC3B or mRFP-EGFP-LC3B fusion proteins as previously reported38 (link),39 (link) and cultured in the presence of 800 μg/ml of geneticin (G418 disulfate salt, Sigma-Aldrich, A1720).
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