The pcDNA3.1 vector for human CypD-WT, CypD-C82S, CypD-C104S, CypD-C157S, and CypD-C203S mutant plasmids was constructed by Sangon Biotech (Shanghai, China). When H9c2 cells grew to approximately 50–60% confluence, the cells were treated with jetPEI TM reagent (Polyplus-transfection, France) before treatment.
Jetpei reagent
Manufactured by Polyplus Transfection
Sourced in France, United States
JetPEI is a cationic polymer-based transfection reagent. It is formulated to facilitate the delivery of nucleic acids, such as plasmid DNA, into mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Dual luciferase reporter assay system, by Promega (6 mentions)
Polybrene, by Merck Group (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Lonza (5 mentions)
Puromycin, by Merck Group (4 mentions)
Dmem glutamax, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Corning (3 mentions)
Penicillin streptomycin, by Harvard Bioscience (3 mentions)
Sh control, by Horizon Discovery (3 mentions)
Pmdlg rre, by Addgene (3 mentions)
Pmd2 g, by Addgene (3 mentions)
Prsv rev, by Addgene (3 mentions)
L glutamine, by Lonza (3 mentions)
Poly l lysine (pll), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Biowest (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions)
Fugene hd, by Promega (3 mentions)
137 protocols using jetpei reagent
1
Plasmid Construction and Transient Transfection
2
Transfection and Luciferase Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfections were performed using previously published methods44 (link). Cultures were grown to subconfluence in antibiotic-free media (10% FCS). Each transfection contained a pGL3-MMP promoter construct (1 μg), 1.5 μl jetPEITM reagent (Polyplus Transfection, Illkirch, France), and 10 ng phRL-SV40, encoding the internal standard Renilla luciferase. Following transfection, cells were treated with: IL-6, soluble IL-6Rα (Peprotech), PMA and/or human IFN-γ. Luciferase activity was recorded using the BMG FLUOstarOPTIMA microplate reader (BMG Labtech, Melbourne, Australia) using Renilla luciferase as a normalization standard.
3
Regulation of STI-1 Gene Promoter by FOXC1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A fragment containing the 5'-flanking region (~2000 bp) of the human STI-1 gene promoter (NCBI Accession number: NC_000011.9) was generated from human genomic DNA by PCR. This product was cloned into the BamHI and XbaI sites of the pGL3-basic vector (Promega, USA), which contained one putative FOXC1-responsive element (FRE) (5'-CAAGCAAATA -3', -181 to -172), and the generated plasmid was designated pSTI-1-luc1. One additional STI-1 promoter construct (pSTI-1-luc2) using the same downstream primer as for pSTI-1-luc1 did not contain the FRE. In the pSTI-1-mutFRE construct, the putative FRE of pSTI-1-luc1 was replaced from 5'- CAAGCAAATA -3' to 5'-GCGGGTAGGT -3' using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, USA). All constructs were verified by DNA sequencing. NIH3T3 cells at approximately 90% confluence in 24-well plates were transiently co-transfected with reporter plasmid (0.5 μg) and FOXC1 expression vector (pcDNA4-FOXC1) using jetPEITM reagent (Polyplus-Transfection, USA) according to the manufacturer's directions. To correct for variable transfection efficiency, cells were cotransfected with the pRL-SV40 vector (0.05 μg) encoding the Renilla luciferase gene. Luciferase activity of cell lysates was determined with a multiwell luminescence reader (Molecular Devices, USA) using the Dual-Luciferase Reporter Assay System (Promega, USA).
4
Silencing Nedd8 in HepG2 Xenograft Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5×106 HepG2 cells were injected into 10 male athymic C57BL/6J nude mice, as described in Supplemental Material . One week after cell inoculation, animals were divided into 2 experimental groups: (i) siControl (n=5) and (ii) siNedd8 (n=5), 50 μM-siRNA dose were intraperitoneally injected thrice a week using jetPEITM reagent (Polyplus), following manufacturer's instructions.
5
Lentiviral Transduction of K562 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exponentially growing HEK-293T cells were transfected with jetPEI-TM reagent (Polyplus-Transfection) with the above vectors plus the two packaging plasmids psPAX2 and pMD-VSVG to produce the lentiviral pseudo-particles (www.lentiweb.com ). For each virus (Sox6, or EV), 72 h after transfection, the supernatant containing the recombinant particles was collected, filtered and centrifuged at 20,000 g for 8 hours at 4 °C. The viral pellet was re-suspended in PBS and stored in aliquots at −80 °C. Lentiviruses were titrated on HEK-293T cells by measuring the percentage of eGFP+ cells by flow cytometry (see below). K562 transduction was performed overnight at a multiplicity of infection (MOI) equal to 30. Cell were collected and analyzed 72 h after transduction.
6
NF-κB Inhibition via IκBαM Transfection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2×106 Huh7/NF-κB-tk-luc2/rfp cells seeded into 10 cm were transfected with the IκBα mutant vector (p-IκBαM; Clontech) using the protocol provided by jetPEITM reagent (Polyplus Transfection, NY, USA) for NF-κB inhibition. Briefly, 8 μg DNA and 16 μL jetPEI diluted with 500 μL of 150 mM NaCl were mixed and incubated at room temperature for 20 min prior to transfection. BLI and electrophoretic mobility shift assays (EMSA) were performed to confirm the inhibitory effect of the IκBα mutant vector on NF-κB activity.
7
Transfection of STAT3 Mutants in H9c2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human STAT3 wild-type (WT) and cysteine (Cys, C)-to-serine (Ser, S) mutant (C108S, C251S, and C259S) plasmids were constructed using BGI-Write (China). H9c2 cells were transfected with plasmids using jet-PEITM reagent (101-40 N, Polyplus-transfection, France).
8
Generating ADAR-1 Overexpressing Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate ADAR-1 p110 and p150 overexpressing cell lines, lentiviral particles pseudotyped with the VSV-G protein were produced by co-transfecting HEK293T cells in 10cm dishes with 5 μg pLentiCMVPuroDEST vector (Addgene #17452), 2 μg VSV-G Env expression vector pMD2.G (Addgene #12259) and 2 μg Gag-Pol expression vector psPAX2 (Addgene #12260) using Jet PEI reagent (Polyplus #101000020) according to the manufacturer’s instructions. Viral supernatants were harvested 48 h after transfection, filtered (0.45 μm), and stored at −80°C or used directly for transduction. Huh7.5 and HEK293T transduced cell lines were selected in 1 μg/mL or 3.5 μg/mL of puromycin respectively for at least 5 days prior to use in assays. Thereafter, protein lysates were collected from the transduced cells and protein levels of the ADAR-1 p110 and p150 were assessed by immunoblotting.
9
Transfection of Multiple Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Huh7.5, HEK293T, A549, NIH-3T3, MCA-RH 7777, FEA, CRFK, and Vero cells were transfected with Jet Pei reagent (Polyplus #101000020) using 1 μg of plasmid DNA and according to the manufacturer’s instructions. I/1Ki cells were transfected with Lipofectamine 3000 (Invitrogen #L3000008) using the reverse transfection method. 1.5 μL Lipofectamine 3000 was diluted in 25 μL of Opti-MEM Medium (Gibco #31985–062), and 1 μg of plasmid DNA was diluted with 2 μL P3000 reagent in 25 μL of Opti-MEM. Both solutions were mixed, incubated for 15min at room temperature (RT) and added to 1.5x105 cells suspended in 1 mL of culture medium. The cells were kept in suspension with the transfection mix for 15 to 30 minutes RT before being seeded in 24-well plates. Transfection medium was replaced for culture medium 6h post-seeding.
10
Vitamin D Receptor Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested after 48 h of culture, washed once in a preheated growth medium at 37 °C, and resuspended at 5 × 105 cells/mL. Four hundred μL of the cell suspension were transferred to 24-well plates, followed by cotransfection with 0.8 µg VDREx6-Luc luciferase reporter plasmid and 0.2 µg Renilla luciferase (pRL-null) reporter plasmid (internal control) using jetPEI reagent (Polyplus Transfection, Illkrich, France). Four hours later, cells were preincubated for 24 h with either a vehicle, 10 µM TRE-ODN, or mutant TRE (mTRE)-ODN. Subsequently, they were treated for an additional 48 h with 1 nM 1,25D3, 10 µM CA, or 50 µM MMF, alone or in combination. Firefly and Renilla luciferase activities were then measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. Luminescence was determined using a Turner 20/20 luminometer (Turner BioSystems, Sunnyvale, CA, USA). Data are expressed as firefly luciferase-to-Renilla luciferase ratios (RLU) [22 (link)]. The VDREx6-Luc reporter plasmid was gifted by Dr. David G. Garner (University of California, San Francisco, CA, USA). The pRL-null vector was purchased from Promega (Madison, WI, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!