Cardiomyocytes were seeded at equal number of cells (2.0 × 105 per dish) in 35 mm petri dish and maintained in the absence of antibiotic culture medium for 24 h before transfection. Cardiomyocytes were transfected with scrambled-siRNA or gene-specific siRNA (Santa Cruz Biotechnology, TX, USA) using LipofectamineTM 3000 transfection reagent from Invitrogen (Thermo Fisher Scientific, Scotland, UK). The transfection reagent complex was added to the reduced serum media (GibcoTM Opti-MEMTM, Thermo Fisher Scientific, UK) for 8 h, the transfection was continued for another 24 h in serum-containing normal medium.
Gibco opti memtm
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Gibco Opti-MEM is a modification of Eagle's Minimum Essential Medium (MEM) that is designed to support the growth and maintenance of various cell types, including primary cells and sensitive cell lines. It is a serum-free, low-protein medium that is formulated to minimize the amount of background proteins and growth factors. Opti-MEM is commonly used for transfection, virus production, and other cell culture applications that require a reduced serum environment.
Automatically generated - may contain errors
Lab products found in correlation
Gene specific sirna, by Santa Cruz Biotechnology (1 mentions)
Scrambled sirna, by Santa Cruz Biotechnology (1 mentions)
Four well plate, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Genematrix universal rna purification kit, by EURx (1 mentions)
3 protocols using gibco opti memtm
1
Cardiomyocyte Transfection Protocol
2
Adenovirus-Mediated Gene Delivery to Cardiomyocytes
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Adenovirus vectors containing MuRF1 and PPARα were purchased from Shanghai GenePharma (Shanghai, China). Cardiomyocytes were seeded at equal number of cells (2.0 × 105 per dish) in 35 mm petri dish and maintained in the absence of antibiotic culture medium for 24 h before transfection. The viral solution was diluted with reduced serum media (GibcoTM Opti-MEMTM, Thermo Fisher Scientific, UK) at 1:50. After the cells were transfected with adenovirus vector at 37 °C for 24 h, reduced serum DMEM medium was discarded. Another 2 mL fresh culture medium containing 10% FBS was added to each dish for 48 h with various treatments.
3
Transfection and NMD Inhibition in MCF-7 Cells
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MCF-7 cells were plated (∼2 × 105 cells/well) and grown to 90% confluency in 0.5 mL of medium (MEME, 10% fetal bovine serum, 2 mM glutamine, 1% non-essential amino acids, and 1% penicillin/streptomycin) in four-well plates (Nunc, Roskilde, Denmark). Transfections were made with 1 μg of minigene and 2 μL of low toxicity Lipofectamine (Life Technologies, Carlsbad, CA, United States) in GibcoTM Opti-MemTM (Thermo Fisher Scientific, Waltham, MA, United States). Cells were incubated with 300 μg/mL of cycloheximide (Sigma-Aldrich, St. Louis, MO, United States) for 4 h to inhibit nonsense-mediated decay (NMD). RNA was purified with the Genematrix Universal RNA Purification Kit (EURx, Gdansk, Poland) with on-column DNAse I digestion to degrade genomic DNA that could interfere with RT-PCR.
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