Colon cancer cells (SW480, HCT116 cells/well) were plated in 96-well plates and transiently transfected with 0.4 μg of the empty vector or the constitutively activated 100 nM of negative siRNA, DR3, DR4 or Fas siRNA per well, using a mixture of plasmid and the WelFect-EX PLUS reagent in OPTI-MEM, according to the manufacturer’s specification (WelGENE, Seoul, Korea). The p50 mutant (Vp50A, valine 412 was substituted with Alanine) plasmid was also transfected with WelFect-EX PLUS reagent in OPTI-MEM according to the manufacturer׳s specification (WelGENE, Seoul, Korea). DR3 siRNA seq. 5’-GAAGCCCUAAGUACGGUUAtt; DR4 siRNA seq. 5’-CUCUGAUGCUGUUCUUUGAtt; Fas siRNA seq. 5’ -GAACCCGUGUUUGCAAUCAtt.
Opti mem
Manufactured by Welgene
OPTI-MEM is a cell culture medium designed to support the growth and maintenance of a variety of mammalian cell lines, including primary cells and transfected cells. It is a serum-reduced formulation that provides a nutritional environment for cells. OPTI-MEM is intended for use in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Welfect ex plus reagent, by Welgene (2 mentions)
Tnfr1 sirna, by Santa Cruz Biotechnology (1 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Welfect ex plus transfection reagent, by Welgene (1 mentions)
Triptolide, by Selleck Chemicals (1 mentions)
Dimethyl sulfoxide (dmso), by Corning (1 mentions)
4 protocols using opti mem
1
Colon Cancer Cell Transfection Assay
2
Establishing IL-32α Expression in SW620 Cells
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Human SW620 colon cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). Colon cancer cells were incubated with medium RPMI 1640 containing L-glutamine 1X supplemented with 10% fetal bovine serum (FBS), 1% 10000 U/ml penicillin and 10000 μg/ml streptomycin, at 37°C in 5% CO2 humidified air. All reagents were purchased from Invitrogen (Carlsbad, CA, USA). To establish constitutive expression systems of IL-32α, cancer cells were transfected with the pcDNA3.1 or pcDNA3.1-IL-32α vector using the Lipofectamine™ 2000 (Invitrogen). The transfected cells were selected with an antibiotic for neomycin resistance gene, G418 (800 μg ml, Sigma). G418-resistant cells were screened for 3 weeks, and single cell-expanded clones were obtained by serial dilutions. We confirmed the stable expression of IL32α in SW620 cells using the Western blot. The cells were transiently transfected with TNFR1 siRNA (Santa Cruz Biotechnology) or Control siRNA (Santa Cruz Biotechnology) per well using a mixture of siRNA and WelFect-EX Plus reagent in OPTI-MEM, according to the manufacturer's specification (WelGENE, Seoul, Korea).
3
Generating Stable Cell Lines for IL-32γ Studies
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Cells were transiently transfected with control vector (pcDNA3.1( + )–6×Myc), IL-32γ vector, or ITGAV vector per well, using WelFect-EX PLUS transfection reagent in OPTI-MEM, according to the manufacturer’s specification (WelGENE, Seoul, Korea)7 (link). To generate stable cell lines (control vector and IL-32γ-CD133+ A549 cells), transfected CD133+ A549 cells were cultured in G418 containing growth medium (600 μg/ml) for 4 weeks. G418-resistant colonies were expanded and used.
4
Notch Inhibitor Screening in Cells and Zebrafish
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Natural compounds (#L1400) and FDA-approved chemicals (#L1300), including triptolide and DAPT for the Notch inhibitor screening test, were purchased from Selleckchem, (Houston, TX, USA). DMSO was purchased from Corning (USA, Manassas, VA, USA). Pyridone 6 (P6) was purchased from R&D Systems (a Bio-Techne Brand, Minneapolis, MN, USA). In cell-based experiments, P6, DAPT, P6 + DAPT, TP, or DMSO were treated for a selective concentration of 50 nM, 100 nM, 200 nM, 500 nM. The concentrations of each signal inhibitor and TP were determined through referencing their use in various cells [39 (link),40 (link),41 (link)] and zebrafish [74 (link),75 (link)]. The concentration ranges typically used in cell-based experiments to test anticancer agents was applied [76 (link)]. They were incubated at 37 °C, 5% CO2, for 24 h. To reduce error, chemicals were diluted with Opti-MEM (Welgene, Republic of Korea) before suspension. In zebrafish experiments, 24 hpf embryos were placed in 90ϕ Petri dishes filled with 10 mL of E3 buffer (with 0.001 M methylene blue). Embryos were treated with DMSO for the negative control or a 500 nM concentration of P6, DAPT, P6 + DAPT, or triptolide. They were incubated for 72 h in an incubator at 28.5 °C.
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