The miR-224 promoter region −1841/+1035 (relative to the pre-miR sequence was amplified by PCR from HepG2 genomic DNA with the following primers: luc-miR-224_FOR 5′-AATCCTGTGCACCTCATCCTCTGT-3′; luc-miR-224_REV 5′-GACGAGCGGAG AAGGTTCTT-3′. The amplified region was cloned into the pGL3 Basic Vector (Promega, Madison, WI, USA) using the T4 DNA ligase (Promega, Madison, WI, USA) and the construct was confirmed by sequencing. Putative miR-224 seed sequences in the HBV genome (genotipe A; ADW) were amplified by PCR with specific primers (Additional file 1 : Table S6), cloned in the 3′ UTR of the Renilla luciferase gene in the pRL-TK vector (Promega) and confirmed by sequencing. For luciferase assay, cells are transfected with the indicated luciferase reporter constructs and expression vectors using the Lipofectamine Plus reagent (Invitrogen Inc., Carlsbad, CA, USA). Luciferase activity in cell lysates is measured using the Luciferase Assay System (Promega, Madison, WI, USA) 30 h after transfection and the results normalized for protein levels (Bradford, Bio-Rad Laboratories, Hercules, CA, USA). Pre-miRNA-224 and anti-miRNA-224 (Ambion #AM17100 and #AM17000) are transfected with the Lipofectamine Plus reagent (Invitrogen Inc., Carlsbad, CA, USA) at a final concentration of 30 nmol/l and cells were analyzed 30 h after transfection.
Lipofectamine plus reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Italy
Lipofectamine Plus reagent is a transfection reagent used for the delivery of nucleic acids, such as DNA or RNA, into a variety of cell types. It facilitates the uptake of these molecules into cells, enabling gene expression studies, gene silencing, or other applications that require the introduction of foreign genetic material.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (21 mentions)
Streptomycin, by Thermo Fisher Scientific (13 mentions)
Penicillin, by Thermo Fisher Scientific (12 mentions)
Dual luciferase reporter assay system, by Promega (9 mentions)
Opti mem, by Thermo Fisher Scientific (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Lipofectamine, by Thermo Fisher Scientific (7 mentions)
Opti mem medium, by Thermo Fisher Scientific (6 mentions)
Rnaimax, by Thermo Fisher Scientific (5 mentions)
Polybrene, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Biowest (5 mentions)
Puromycin, by Merck Group (5 mentions)
Polyethylenimine (pei), by Polysciences (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Dual glo luciferase assay system, by Promega (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Ecl solution, by Cytiva (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Protein g sepharose beads, by Cytiva (4 mentions)
276 protocols using lipofectamine plus reagent
1
Characterization of miR-224 Promoter and Target
2
Fluorescent Fusion Protein Transfection for DAPK1 and Cytoskeletal Studies
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Transfections were carried out 1 day before measurements using Lipofectamine Plus Reagent (Invitrogen) according to the manufacturer’s instructions. Expression vectors encoding the following fluorescent fusion proteins were used: EGFP-DAPK1 WT, EGFP-DAPK1 K42A, mCherry-DAPK1 WT, mCherry-DAPK1 K42A, Emerald-Tpm1.1 WT, Emerald-Tpm1.1 S283E (phosphomimetic mutant), Emerald-Tpm1.1 S283A (non-phosphorylated mutant), mCherry-paxillin, GFP-talin1 WT, mRuby-talin1 head, mCherry-talin1 rod, GFP-non-cleavable talin1, EGFP-DAPK1-DYD (phosphomimetic mutant), EGFP-DAPK1-DYF (non-phosphorylated mutant), EGFP-DAPK1-DYF-K42A (non-phosphorylated inactive mutant), mCherry-PTPN12. MEFs were seeded into a 6-well dish on day 0 and transfected with control shRNA (Sigma) or mouse DAPK1 shRNA (Sigma) using Lipofectamine Plus Reagent (Invitrogen) on day 1, followed by selection in 2 μg/ml puromycin for 72 h. Control siRNA, mouse Src siRNA and mouse PTPN12 siRNA (Santa Cruz) were transfected using siRNA transfection reagent (Santa Cruz).
3
Assay for CPI-17 Promoter Activity
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Rat aortic VSMCs were cotransfected with pGL3-CPI-17 firefly luciferase vectors and a Renilla luciferase control vector (pRL-Null; Promega) using Lipofectamine-Plus reagent (Life Technologies). After TNF treatment (10 ng/ml, 48 h), CPI-17 promoter activity was assayed by a modified dual-luciferase enzyme assay as described previously (25 (link), 26 (link), 47 (link)).
4
Cell Line Cultivation and Transfection
5
Lentiviral Transduction of Stem Cell Factors
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Using the Lipofectamine-Plus reagent (Life Technologies), pMXs-green fluorescent protein (GFP), pMXs-OCT4, pMXs-SOX2, pMXs-KLF, and pMXs-c-MYC were transfected into prepared PLAT-GP cells (2.5 × 104 cells/cm2) with gag-pol and VSV-G [17 (link)]. At 4 h after transfection, the medium was replaced with DMEM with 10% FBS. After 48 and 72 h, the supernatant was collected and filtered through a polyvinylidene fluoride (PVDF) filter (pore size 0.45 μm; EMD Millipore, Billerica, MA, USA). Finally, it was concentrated with a Retro-X concentrator (Clontech Laboratories, Inc., Mountain View, CA, USA) for 24 h based on the protocol provided by the manufacturer. Briefly, the Retro-X was added to the acquired supernatant containing virus in a 1:3 volume ratio. Then the mixture was incubated at 4 °C for a day. After a day they were centrifuged at 1500 × g at 4 °C for 45 min. After removing supernatant we added FGM-2. The volume of FGM-2 added was 1/10 of the original solution during centrifugation.
6
Regulation of NF-κB Signaling by TLR4 and MD-2 in HEK293 Cells
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Human embryonic kidney 293 (HEK293) cells were seeded in 96-well plates at 2 × 104 cells per well and incubated overnight in a 5% CO2 incubator at 37°C. In the morning, cells were transiently transfected with 50 ng TLR4 expression vector (pcDNA3.1-hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid #13086) or empty vector, along with 15 ng of Firefly luciferase NF-κB reporter vector (pNifty-Luc, InvivoGen), 15 ng of Renilla luciferase control vector (pRL-TK, Promega), 10 ng of wt or mutant MD-2 expression vector (pFlag-CMV1-hMD2), and 10 ng of CD14 expression vector (pCDNA3.1-hCD14, a gift from Doug Golenbock, Addgene plasmid #13645) per well using Lipofectamine Plus reagent (LifeTechnology). Four h after transfection, cells were incubated 24 h in media containing 10% fetal bovine serum (FBS). Twenty-four hours after transfection, cells were incubated with media with 1% FBS (control), or media with 1% FBS supplemented with heme (10 μM), LPS (10 ng/ml, Escherichia coli, serotype O111:B4; Sigma-Aldrich) or heme + LPS for 6 h, then cells were lysed and luciferase activity was measured.
7
Luciferase Reporter Assays and Transfection Optimization
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Transfection and luciferase reporter assays were performed as described (10). Transient transfection assays were performed using Lipofectamine Plus reagent (Life Technologies). For the luciferase reporter assay shown in Fig. 1H , Saos2 cells were seeded in 96-well dishes and co-transfected with 60 ng of a firefly luciferase reporter gene and 3 ng of each p53 gene cloned in the pcDNA3 vector together with 15 ng of a Renilla luciferase expression vector (pGL4 [TK-Rluc] vector, Promega) as an internal control for transfection efficiency. For the luciferase reporter assay shown in Fig. 3 , H1299 cells were co-transfected with 30 ng of a firefly luciferase reporter gene and 5 ng of each IER5 gene cloned in pcDNA3 vector together with 3 ng of a Renilla luciferase expression vector pGL4 [TK-Rluc] vector. Cells were harvested 48 hrs (Fig. 1H ) or 24 hrs (Fig. 3 ) post-transfection, and analyzed using the Dual-Luciferase Reporter Assay System (Promega). All of the luciferase reporter assay data are the mean-fold activation +/− SD of three independent experiments. The siRNAs were introduced using RNAiMAX (Invitrogen, Carlsbad, CA). Control, ON-target plus IER5-targeting, HSF1-targeting, p53-targeting and PPP2CA-targeting siRNAs were purchased from Dharmacon Research (Lafayette, Colorado).
8
Stable Transfection of Ob-Rb Receptor
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The human Ob-Rb leptin receptor construct was a gift from Genetech Inc. The construct was transfected into SH-SY5Y cells using LipofectAMINE PLUS Reagent (Life Technologies Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. After the transfection, stable transfectants were obtained by selection with the antibiotic G418.
9
Lentiviral Transduction for Gene Expression
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The delivery of expression constructs cells was through lentiviral infection. Viruses were generated in 293T cells. To produce virus, plasmids including the lentiviral shRNA vector, pCMVR8.74, and pMD2.G were cotransfected into 293T cells using LipofectAMINE Plus reagent from Life Technologies (Carlsbad, CA) according to the instruction. At 48 h post‐transfection, virus‐containing supernatants were collected and centrifuged at 3000 g for 5 min to remove suspended target cells. The supernatants were mixed with polybrene at final working concentration of 10 μg/mL for infection. Medium containing virus was replaced with fresh growth medium 6 h after viral infection. The cells were used for experiments 2–3 days post‐transduction.
10
Measuring STAT1 and STAT6 Activation
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Cells at 40% to 50% confluence in the 12-well plates were transfected with a reporter luciferase plasmid containing the interferon stimulated response element (ISRE) (pGL4.45[luc2P/ISRE/Hygro]; Promega) and containing four tandem copies of the STAT6 binding site (p4xSTAT6- Luc2P; Addgene, Cambridge, MA, USA). The reporter vector was then transfected into the cell lines with Lipofectamine plus reagent (Lifetechnology) according to the manufacturer's instructions. The total amount of plasmid DNA per well was adjusted to be the same by adding suitable amounts of empty vector. To activate the STAT1 and STAT6 reporter vectors, IL-4 (10 ng/ml) and IFN-γ (10 ng/ml) were used to treat to the cells for 6 h. Cells were harvested 48 h after transfection, and luciferase activity was measured using a commercial luciferase assay kit (Promega) on the TD20/20 Turner luminometer (Turner Design Instruments, Sunnyvale, CA, USA). The luciferase activity of each sample was normalized to that of the corresponding sample transfected with pGL4. All experimental and control groups contained at least three wells, and the results were reported as mean absorption±standard error.
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