EA.hy926 cells, a human endothelial cell line, were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics at 37 °C in 5% CO2 and 95% room air. For cellular assays, we switched the cells to DMEM containing 1% FBS. Next, 80% confluent cells were stimulated with the indicated dose and time of juglone, BAY11-7082 or vehicle, prior to incubation with oxLDL (50 μg/mL) for 15 min or 24 h. Likewise, we transfected the cells with 100 nM of siRNA (all from Genomeditech, Shanghai, China) targeting Pin1 or with scrambled siRNA using the riboFECT™ CP regent (RiboBio Co., Ltd., Guangzhou, China). The cells were stimulated with oxLDL (50 μg/mL) 48 h after siRNA transfection.
Ribofect cp regent
Manufactured by RiboBio
Sourced in China
RiboFECT™ CP is a cationic polymer-based transfection reagent for delivering nucleic acids, such as plasmid DNA, siRNA, and miRNA, into a variety of mammalian cell lines. It is designed to efficiently complex and protect nucleic acids, facilitating their cellular uptake and intracellular delivery.
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Lab products found in correlation
4 protocols using ribofect cp regent
1
Endothelial Cell Oxidative Stress Response
2
AMPK Knockdown via siRNA Transfection
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AMPK siRNA was synthesized by Generay Biotech (Shanghai, China). Cells were transiently transfected using riboFECT CP Regent (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. The sequence of si-AMPK: sense: 5′-GUUGCCUACCAUCUCAUAAUATT-3′; antisense: 5′-UAUUAUGAGAUGGUAGGCAACTT-3′; AMPK inhibitor Compound C was phased from MedChemExpress.
3
Silencing TREM-1 in Microglia
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Negative control siRNA (UCUCCGAACGUGUCACGUT) and TREM-1 siRNA (CCCAGTGACACAACTACAA) were synthesized by RIBOBIO (China). siRNA (20 μM) of 1.25 μl and riboFECTTM CP Regent (RIBOBIO, China) of 3 μl were mixed in 30 μl riboFECTTM CP Buffer to generate a transfection mixture. Then the mixture was added into DMEM (siRNA concentration: 50 nM) and cultured with microglia for 48 h. Real-time polymerase chain reaction (PCR) and western blot analysis were performed to examine the transfection efficiency.
4
Drosophila S2 Cell Line Transfection
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Drosophila S2 cell line was cultured in Schneider’s medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Thermo Fisher Scientific). The cells were plated in a 24-well plate and incubated for 24 h. Small interfering RNA (LBR: CGAAGACAATCTCAAATCT) and negative control were transiently transfected using riboFECTTM CP Regent (RIBOBIO C10511-05) according to the protocol provided by the manufacturer. Plasmids were transiently transfected using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) according to the protocol provided by the manufacturer. Expression of lbr, mts, and dbt under pMT promoter was induced by adding 500 μM CuSO4 to the culture upon the completion of transfection and incubation for another 48 h unless specified otherwise. For experiments in which cycloheximide [10 mg/ml, MedChemExpress (MCE)], Cal A (30 nM, MCE), okadaic acid, sodium salt (5 nM, Sigma-Aldrich), and D4476 (10 or 20 μM, MCE) were used, they were added at the indicated time post transfection.
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