Culturing and Transfecting Endothelial and Kidney Cells

Human umbilical vein endothelial cells (HUVECs) were grown on 0.1% gelatin coating in EndoGRO basal medium (Millipore) containing 5% fetal bovine serum (FBS) as previously described30 (link). Passage number did not exceed P7. Human epithelial kidney cells (HEK293) were used in promoter activity experiments. HEK293 were grown in 10% FBS DMEM media. Human TNFα (R&D Systems) was used in all in vitro experiments at 10ng/ml. Protein knockdown by siRNA (Ambion) in HUVECs was done by Oligofectamine (Invitrogen) for 3h and followed by 48h growth. Transfection of HEK293 by plasmid DNA was accomplished through Polyfect (Qiagen) according to manufacturer protocol. Transfection was allowed for 18h prior to treatment with harvest or TNFα treatment. THP-1 monocytes were grown in RPMI media containing 10% FBS.

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Chen G.F., Sudhahar V., Youn S.W., Das A., Cho J., Kamiya T., Urao N., McKinney R.D., Surenkhuu B., Hamakubo T., Iwanari H., Li S., Christman J.W., Shantikumar S., Angelini G.D., Emanueli C., Ushio-Fukai M, & Fukai T. (2015). Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function. Scientific Reports, 5, 14780.

Publication 2015

EndoGRO basal medium Human TNFα siRNA Oligofectamine Polyfect

Epithelial cells Fetal bovine serum Gelatin Human Human umbilical vein endothelial cells Kidney Monocytes Oligofectamine Plasmid Protein Sirna Tnfα Transfection

Corresponding Organization :

Other organizations : University of Illinois at Chicago, The University of Tokyo, The University of Texas Health Science Center at San Antonio, The Ohio State University Wexner Medical Center, NIHR Bristol Cardiovascular Biomedical Research Unit, University of Bristol

Top 5 similar protocols

Variable analysis

independent variables

Protein knockdown by siRNA (Ambion) in HUVECs
Transfection of HEK293 by plasmid DNA
Human TNFα (R&D Systems) treatment at 10ng/ml

dependent variables

Promoter activity in HEK293 cells

control variables

HUVECs grown on 0.1% gelatin coating in EndoGRO basal medium (Millipore) containing 5% fetal bovine serum (FBS)
HEK293 cells grown in 10% FBS DMEM media
THP-1 monocytes grown in RPMI media containing 10% FBS
Passage number of HUVECs did not exceed P7

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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