BV2 cells (2 × 105 cells/well on a 12-well plate) were transfected with 1 μg of reported plasmid DNA using Metafectene transfection reagent (Biontex, Martinsried/Planegg, Germany). The effect of PAP on reporter gene activity was determined by pre-treating the cells with PAP prior to stimulation with LPS (100 ng/mL) for 6 h. After preparing the cell lysates, the luciferase assay was performed as previously described [36 (link)].
Metafectene transfection reagent
Manufactured by Biontex
Sourced in Germany, United States
Metafectene is a transfection reagent designed for efficient delivery of nucleic acids, such as DNA and RNA, into eukaryotic cells. It facilitates the uptake of genetic material by the target cells, enabling various applications in cell biology and molecular genetics research.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (2 mentions)
Pierce protein a g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Pierce ip lysis, by Thermo Fisher Scientific (1 mentions)
Pierce anti c myc magnetic beads, by Thermo Fisher Scientific (1 mentions)
Endo h, by New England Biolabs (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Supersignal west pico chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 l glutamine, by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Anti rabbit igg w401b, by Promega (1 mentions)
Anti mouse igg w402b, by Promega (1 mentions)
Sb431542, by Bio-Techne (1 mentions)
Pcdna3 vector, by Thermo Fisher Scientific (1 mentions)
Activin a 338 ac, by R&D Systems (1 mentions)
C fos, by Merck Group (1 mentions)
S1pr3, by Merck Group (1 mentions)
C jun, by Merck Group (1 mentions)
Pdgfr, by Merck Group (1 mentions)
C src, by Merck Group (1 mentions)
17 protocols using metafectene transfection reagent
1
BV2 Cell Transfection and PAP Effects
2
CD2v Variant Immunoprecipitation and Glycosylation
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About 10 × 106 COS-1 cells were transfected with 1 µg/1 × 106 cells of the specific vector using Metafectene transfection reagent (Biontex). At 6 hpt, cells were infected with VV-T7 (MOI = 0.5 PFU/mL). At 16 hpi, cells were collected and lysed with 1 mL of Pierce IP Lysis (ThermoFisher Scientific) and protease and phosphatase inhibitors for 2 h on ice. Lysates were centrifuged and precleared with Pierce Protein A/G Magnetic Beads (ThermoFisher Scientific). CD2v variants were immunoprecipitated using either Pierce Anti-c-Myc Magnetic Beads or anti-CD2v bonded to magnetic beads Pierce Protein A/G Magnetic Beads (ThermoFisher Scientific), as indicated, at 4 °C with rotation overnight. Different immunoprecipitates were eluted and non-treated or treated with either endoglycosidases PNGase-F or Endo-H (New England BioLabs). Briefly, IP elution was denaturalized using denaturalized buffer at 100 °C for 10 min and then chilled on ice, centrifuged at 11,000 rpm, and incubated with PNGase-F or Endo-H (1 h at 37 °C). Finally, samples were analyzed by Western blot.
3
Protein Expression in Huh7-T7 and SK6 Cells
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The applied procedures have been described [19 (link), 43 (link)]. Briefly, Huh7-T7 cells, which express T7 RNA-polymerase [64 (link)], or SK6 cells were infected with modified vaccinia virus Ankara-T7pol (MVA-T7pol) [67 (link)], subsequently transfected with 4–6 μg of plasmid DNA by using 10 μl Metafectene transfection reagent (Biontex). Protein expression was carried out for 18 h and cells were further processed.
4
TGF-β Signaling Pathway Regulation
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RPMI1640/L-glutamine, DMEM/High glucose, 2β-mercaptoethanol (35602), antibiotic-antimycotic (SV30079.01) and Supersignal West Pico Chemiluminescence Substrate (34080) were purchased from Thermo Scientific. Sodium pyruvate (25-000-CI) was purchased from Mediatech Inc. Fetal Bovine Serum (FBS) (S01520) was purchased from Biowest. Metafectene transfection reagent (T020) was purchased from Biontex Laboratories (Germany). 3[H]-2-deoxyglucose was purchased from MP Biochemicals. TGF-β1 (240-B), TGF-β3 (243-B3), Nodal (1315-ND-025CF) and Activin A (338-AC) were obtained from R&D Systems. TGF-β2 was kindly provided by Dr. Steven Ledbetter (Genzyme). SB 431542 (#1614) was purchased from Tocris. The 3TP-lux reporter has been previously described (22 (link), 23 (link)). Anti-Smad2 rabbit polyclonal (51-1300), rabbit polyclonal anti-Smad3 (51-1500), were purchased from Zymed. Rabbit polyclonal anti-phosphoSmad3 was a kind gift of Dr. Ed Leof, Mayo Clinic (24 (link)). Mouse monoclonal anti-Smad4 (B8)(sc-7966) and rabbit polyclonal anti-phosphoSmad2 (06-829) were purchased from Santa Cruz Biotechnology and Upstate Biotechnology, respectively. The secondary antibodies anti-rabbit IgG (W401B) and anti-mouse IgG (W402B) were purchased from Promega. The pcDNA3 vector was obtained from Invitrogen.
5
Luciferase Assay for BV2 Cells
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BV2 cells were transfected with 1 μg of reporter plasmid DNA ([κB]3-luc, ARE-luc, CRE-luc, or PPRE-luc) using Metafectene transfection reagent (Biontex, Martinsried/Planegg, Germany). The effect of GTS-21 on reporter gene activity was assessed by pre-treating the cells with GTS-21 before LPS stimulation (100 ng/mL; 6 h) after 36 h transfection. Following that, cell lysates were collected and a luciferase assay was performed as described previously [29 (link)].
6
siRNA-Mediated Silencing of Key Signaling Proteins
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Human siRNAs of PKCδ (SASI_Hs01_00061170), PYK2 (SASI_Hs01_00032249), p47phox (SASI_Hs02_00302212), p65 (SASI_Hs01_00171091), or scrambled control (St. Louis, MO, USA) were used to knockdown the specific gene expression. The transient transfection of siRNA (100 nM) was performed with Metafectene transfection reagent according to the manufacturer’s protocol (Biontex, Germany).
7
Targeted Gene Silencing by siRNA
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Human siRNAs of scrambled, S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, and c-Fos were from Sigma (St. Louis, MO). Transient transfection of siRNAs was carried out using Metafectene transfection reagent (Biontex, Germany). siRNA (100 nM) was formulated with Metafectene transfection reagent according to the manufacturer’s instruction.
8
Cell-to-Cell Transmission of HIV-1
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SupT1 or primary CD4+ T cells were infected with cell-free virus as described (Lepelley et al., 2011 (link)). For the virus cell-to-cell transmission assay utilizing SupT1 as donors, 5 × 106 cells were first infected with 100–200 ng NL4-3 and cultured for 2 days to achieve 30%–50% Gag+ cells. Cells were then induced or not induced for IFITM. A total of 1 × 105 donor cells was mixed with 3 × 105 target SupT1 cells labeled with CellTrace Far Red DDAO-SE (Life Technologies) in 24-well plates. After 2 hr, 2× 105 target (Far Red+) cells were collected and separated from donors using a FACSAria cell sorter and plated in a 96-well plate. Cells were then analyzed by flow cytometry. When 293T cell were used as donors, 1 × 105 cells were cotransfected in a 24-well plate with the pNL4-3 provirus (1 μg) and FLAG-IFITM (1 μg, unless otherwise stated) using Metafectene transfection reagent (Biontex). A total of 3 × 105 labeled SupT1 targets (∼1:3 donor:target) were added 48 hr later. After 2 hr of coculture, targets were manually separated from donors and transferred to a 48-well plate. To measure infectivity, viral particles were purified on a sucrose cushion, and the inoculum (adjusted for p24 content) was added to fresh SupT1 cells. Unless otherwise stated, 25 ng of p24 was added to 105 cells. Infection was assessed by measuring the fraction of Gag+ cells after 2–3 days.
9
Adenoviral DNA Linearization and Transfection
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To ensure the replication and packaging efficiency of pAd-BMP2, the plasmid was linearized with PacI and purified successively using phenol, chloroform and isopentanol. After centrifugation, the supernatant was mixed thoroughly with ethanol to precipitate DNA, and the pellet was washed and dissolved in dH2O. HEK293 cells (American Type Culture Collection, USA) were transfected with linearized adenoviral DNA using METAFECTENE™ transfection reagent (Biontex, Germany). HEK293 cells were observed for cytopathic effect (CPE) for 7 days post-transfection. When more than 50% cells had detached from the bottoms of culture flasks, the cells were collected, subjected to three freeze-thaw cycles, and stored at −74°C.
10
Regulation of ATR, ATM, and PCNT Kinases
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hTERT-immortalized fibroblasts were control (1BR3hTERT), ATR (FO2-98hTERT), ATM (AT1BRhTERT) and PCNT (ASBhTERT). siRNA was carried out using the appropriate Smartpool (Dharmacon, Lafayette, CO, USA) and Metafectene Transfection Reagent (Biontex, Munich, Germany). The ATR inhibitor, VE-821 (Selleckchem/Stratech, Newmarket, UK), was used at 10 µm . The Aurora A kinase inhibitor, MLN8237 (Selleckchem/Stratech), was used at 0.5 µm . The Polo-like kinase 1 inhibitor, BI2536 (Axon Medchem, Groningen, The Netherlands), was used at 100 nm . The Chk1 inhibitor, UCN-01 (Merck, Kenilworth, NJ, USA), was used at 600 nm .
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