Human Raji CD19+ Burkitt’s lymphoma cells were cultured in R10 media, and subsequently the cells were collected by centrifugation and re-suspension. A total of 2 μg of the pGL4.51 plasmid (luc2/CMV/Neo; Promega E1320, Madison, MI, USA) per 2×106 cells was electrotransfected using the T-021 program with Amaxa Nucleofector II Technology (Lonza). After culture in R10 media in the presence of 800 μM G418 (Invitrogen, Carlsbad, CA, USA) for ~2 weeks, the Raji cells showed the stable expression of a firefly luciferase fusion protein. CAR-modified T cells were cultured in 5% FBS-X-VIVO 15 media. For transient expression in activated CAR-modified T cells, the cells were suspended with 1 μg of modified mmRNA in 100 μl of the human T-cell Nucleofector Kit (Lonza) and subsequently applied to the T-023 program equipped with Nucleofector Technology.
Luc2 cmv neo
Manufactured by Promega
Sourced in United States
The Luc2/CMV/Neo is a vector designed for the stable expression of reporter genes in mammalian cells. It contains the firefly luciferase gene (Luc2) under the control of the cytomegalovirus (CMV) promoter and the neomycin resistance gene (Neo) for G418 selection.
Automatically generated - may contain errors
Lab products found in correlation
Human t cell nucleofector kit, by Lonza (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase system, by Promega (1 mentions)
Nlucp nf κb re hygro, by Promega (1 mentions)
Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (1 mentions)
Fugene transfection reagent, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
A2780, by Merck Group (1 mentions)
Victor luminometer, by PerkinElmer (1 mentions)
A2780, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
5 protocols using luc2 cmv neo
1
Stable Raji Cell Line for Luciferase Assay
2
Dual-Luciferase Assay for NF-κB Activity
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Human SW982 cells were simultaneously transfected with siRNA, the pNL3.2 (NlucP/NF-κB-RE/Hygro; Promega, Madison, WI) vector, and the pGL-CMV (luc2/CMV/Neo; Promega) vector using Lipofectamine 3000 (Thermo Fisher Scientific). Thirty-six hours after transfection, cells were serum-starved for 12 h and then stimulated with LPS (1 μg/mL) for 6 h. Lysates were prepared and analysed using the Dual-Luciferase System (Promega).
3
Stable Luciferase-Expressing HCC1954 Cells
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Stable luciferase-expressing HCC1954 cells (HCC1954-luc) were generated by transfection with the firefly luciferase gene (luc2) encoding plasmid pGL4.51[luc2/CMV/Neo] (Promega) using the Lipofectamine 3000 Transfection Kit (Thermo Fisher Scientific). For the selection of transfected cells, 200 µg/ml geneticin disulfate (G418) (Carl Roth) was used.
4
Orthotopic and Metastatic Cancer Models
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We constructed an orthotopic model of hepatocellular carcinoma cells and a metastatic model of colon cancer cells. Tumor engraftment and tumor growth in the liver were confirmed with in vivo bioluminescent imaging (IVIS Lumina; Xenogen) as previously described.19 Luciferase expressing HepG2‐luc human hepatocellular carcinoma cells were created by transfecting pGL4.51 (luc2/CMV/Neo; Promega) with FuGENE transfection reagent (Promega) according to the manufacturer's protocol. The orthotopic model was constructed by inoculating HepG2‐luc cells (1 × 106 cells), into the liver of SCID mice. After the confirmation of tumor engraftment, 2',4'‐BNA/LNA VASH2‐ASO2 were diluted in autoclaved distilled water, and were injected i.p. every 3 days for 18 days at the dose of 10mg/kg.
Luciferase expressing human colon carcinoma cells were obtained from the Japanese Collection of Research Bioresources Cell Bank. The metastatic model was constructed by injecting HT29‐luc cells (1 × 106 cells), into the spleen of nude mice. Three days after inoculation, 2',4'‐BNA/LNA VASH2‐ASO2 were diluted in autoclaved distilled water, and were injected i.p. every 3 days for 18 days at the dose of 10mg/kg.
The liver tissue was obtained for ex vivo imaging and histological analysis. Histological analysis was undertaken with H&E staining using standard procedures.
Luciferase expressing human colon carcinoma cells were obtained from the Japanese Collection of Research Bioresources Cell Bank. The metastatic model was constructed by injecting HT29‐luc cells (1 × 106 cells), into the spleen of nude mice. Three days after inoculation, 2',4'‐BNA/LNA VASH2‐ASO2 were diluted in autoclaved distilled water, and were injected i.p. every 3 days for 18 days at the dose of 10mg/kg.
The liver tissue was obtained for ex vivo imaging and histological analysis. Histological analysis was undertaken with H&E staining using standard procedures.
5
Establishment and Characterization of Ovarian Cancer Cell Lines
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The human ovarian cancer cell lines used in this study were the adenocarcinoma SKOV3, Caov-3, and OVCAR-3 (Korea Biotechnology Commercialization Center, KBCC; Incheon, Korea); A2780 (Sigma-Aldrich, St. Louis, MO, USA), and clear cell carcinoma JHOC-5 (Rikagaku Kenkyujyo, RIKEN, Tsukuba, Japan) cell lines. Cell culture media were obtained from Thermo Scientific Hyclone (Waltham, MA, USA). OVCAR-3, SKOV3, and A2780 cells were maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Scientific Hyclone). Caov-3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. The JHOC-5 cells were maintained in DMEM/F12 medium supplemented with 10% FBS. All cells were grown at 37°C in a humidified incubator.
A2780 cells overexpressing firefly luciferase were generated as previously described (24 (link)). Briefly, A2780 cells were transfected with the firefly luciferase reporter plasmid pGL 4.51 (luc2/CMV/Neo; Promega, Madison, WI, USA) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). The cells were cultured in medium containing 100 μg/ml G418 to select positive clones. The expression of luciferase was determined using a Dual-Luciferase reporter assay system (Promega) and luminescence was measured using a Victor luminometer (Perkin Elmer, Waltham, MA, USA).
A2780 cells overexpressing firefly luciferase were generated as previously described (24 (link)). Briefly, A2780 cells were transfected with the firefly luciferase reporter plasmid pGL 4.51 (luc2/CMV/Neo; Promega, Madison, WI, USA) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). The cells were cultured in medium containing 100 μg/ml G418 to select positive clones. The expression of luciferase was determined using a Dual-Luciferase reporter assay system (Promega) and luminescence was measured using a Victor luminometer (Perkin Elmer, Waltham, MA, USA).
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