Cells were cultured in DMEM (Life Tech.) supplemented with 8% fetal bovine serum (FBS), penicillin and streptomycin. FLCN-HA, EGFP-PAT1 or LAMP1-EGFP plasmid was introduced into HEK293 cells using the TurboFect transfection reagent (Life Tech.). Stable cell lines were selected on the bases of the neomycin resistance. For RNAi experiments, RNAiMax diluted in OptiMEM (Life Tech.) was used to deliver siRNA according to the manufacturer's instructions; shRNA sequences were cloned into the expressing plasmid (pCD513B-U6) and co-transfected with the helper plasmids (GAG, REV and VSV-G) into HEK293T cells using TurboFect transfection reagent (Life Tech.). Purified and concentrated virus was used to infect cells with puromycin selection. To confirm the data of the knockdown experiments, we tested at least two RNAi of each target gene following published works and obtained similar results, including two siRNAs of FLCN (siFLCN-1 and 2 in ref. 21 (link)), two shRNAs of FLCN (shFLCN-1 and 2 in ref. 29 (link)), two siRNAs of PAT1 (si158 and si160 in ref. 30 (link)) and two siRNAs of PAT4 (si435 and si437 in ref. 30 (link)). After transfection, cells were allowed to recover for at least 36 hrs before the following treatment.
Turbofect transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, Lithuania, Canada, Italy, United Kingdom, Jamaica, Denmark
TurboFect is a cationic polymer-based transfection reagent used for the delivery of nucleic acids, such as plasmid DNA, siRNA, and oligonucleotides, into a variety of mammalian cell lines. It facilitates the uptake of these molecules by cells, enabling efficient gene expression or gene silencing experiments.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (47 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (31 mentions)
Dual luciferase reporter assay system, by Promega (25 mentions)
Streptomycin, by Thermo Fisher Scientific (21 mentions)
Polybrene, by Merck Group (20 mentions)
Opti mem, by Thermo Fisher Scientific (20 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (20 mentions)
Penicillin, by Thermo Fisher Scientific (18 mentions)
Puromycin, by Thermo Fisher Scientific (15 mentions)
Luciferase assay system, by Promega (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (12 mentions)
Dual glo luciferase assay system, by Promega (12 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (11 mentions)
Puromycin, by Merck Group (9 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (9 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Merck Group (7 mentions)
Dual luciferase assay system, by Promega (7 mentions)
Mg132, by Merck Group (7 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
841 protocols using turbofect transfection reagent
1
Cell Culture and Genetic Manipulation
2
Plasmid Transfection in Cell Lines
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The following plasmids were constructed and purchased from GeneChem (Shanghai, China): plasmids (CMV-MCS-3FLAG-SV40-neomycin) containing the full length of human MLKL coding sequence (gene ID: 197,259) and its mutant form (gene ID: 197,259 (S454A)) or control plasmids; the plasmids (CMV-MCS-Myc-SV40-neomycin) containing the full length of human GJA1 coding sequence (gene ID: 2697) and its mutant form (gene ID: 2697 (K303G)) or control plasmids; the plasmids (CMV-MCS-HA-SV40-neomycin) containing human Ub coding sequence (gene ID: 7316); and the plasmids (CMV-MCS-V5-SV40-neomycin) containing human VHL coding sequence (gene ID: 7428). TurboFect Transfection Reagent (Thermo, Cat# R0531) and plasmids were prepared in the following ratios: plasmid (1 μg): TurboFect Transfection Reagent (3 μL). After 20-minute incubation, the mixture was added to the cells seeded in the dish and left for 48 h for further analysis, with appropriate replacement of fresh medium. The final concentration of plasmid was 0.8 μg/mL.
3
Colonic Cancer Cell Line Transfection
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Colonic cancer cell line SW480 and HT-29 cells and HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium, DMEM (Gibco by life technologies), with 10% Fetal Bovine Serum (FBS) supplemented with 100 U/ml penicillin, 100 U/ml streptomycin and 1% glutamine at 37°C with 5% CO2. Transfections were performed by transfecting plasmids pCMV-Tag2B, pCMV-HBXIP or pCMV-p65 into colonic cancer SW480 or HT-29 cells with TurboFect Transfection Reagent (Thermo Scientific). Cells were collected and seeded in 6-well, 24-well or 96-well plates for 24 hr and then were transfected with plasmids or siRNAs. All transfections were performed with TurboFect Transfection Reagent according to (Thermo Scientific) manufacturer’s instructions. The siRNAs used in our study were as follows: negative control siRNA (si-Control), HBXIP siRNA (si-HBXIP), p65 siRNA (si-p65) and PPARδ siRNA (si-PPARδ) (Shanghai GenePharma Co., Ltd).
4
U2OS Cell Transient Transfection
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Transient transfections of U2OS cells were performed using Turbofect transfection reagent (Fermentas) in Opti-MEM® I Reduced-Serum Medium (Gibco). For the immunofluorescence experiments, cells were plated in six-well plates 4 h before transfection. Transfection reactions were carried out by mixing 4 μg of DNA with 6 μl of Turbofect in a total volume of 400 μl of Opti-MEM. The average transfection efficiency was 40 %.
5
Stable Overexpression of ADPGK-AS1 in THP1 Cells
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Stable overexpression of ADPGK‐AS1 and stable overexpression in THP1 cells was achieved using lentiviral vectors (pLV‐puro‐CMV) containing cDNA for a specific lncRNA that was purchased from VectorBuilder (Chicago, USA). An empty vector was used as a negative control. Lentiviral particles were produced by cotransfection of HEK293T cells with a lentiviral plasmid (psPAX2; Addgene, Watertown, USA) and packaging plasmid (pMD2.G; Addgene, Watertown, USA) using TurboFect transfection reagent (Fermentas, Waltham, USA). The medium containing the viral particles was collected after 48–72 h of transfection. THP1 cells were cultured in 6‐well plates and transduced with lentivirus in the presence of 6 μg/ml polybrene (Merck, Darmstadt, Germany). Thereafter, 24 h after transfection, the medium was replaced with a fresh medium containing puromycin (Gibco, Texas, USA).
6
Transfection of Cells with miRNA and shRNA
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Cells were seeded into a 6-well plate, incubated for 24 hr, and then transfected with plasmid or RNA using TurboFect Transfection Reagent (Fermentas, Hanover, MD) according to the manufacturer's guidelines. The following plasmid and RNAs were used: pLKO. TRC-miR-125a-5p: 5′-UCCCUGAGACCCUUUAACCUGUG 3′-ends, pLKO.1-HDAC4 shRNA-1: 5′-CGACTCATCTTG TAGCTTATT 3′-ends, pLKO.1-HDAC4 shRNA-2: 5′-GAATCTGAACCACTGCATTTC 3′-ends.
7
Construct Transfection and Selection
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The pcDNA3.1 FLAG plasmids construct carrying the PPL CC or GG genotype respectively at position 4717 were purchased from GenScript. Plasmids were transfected into the ORL-153, ORL-214, ORL-115 and Cal27 using Turbofect transfection reagent (Fermentas) according to the manufacturer’s instructions. Control cells were transfected with the pCDNA3.1 FLAG vector. Transfected cells were selected using G418 (Sigma-Aldrich) at 400 µg/ml for 7 days.
8
Immunoprecipitation of GEMC1 protein
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HA-tagged GemC1, Geminin-GFP, Geminin(1–72)-GFP, Idas-GFP and Cdt1-GFP were cloned in pcDNA3.1 for expression in mammalian cells. U2OS cells were cultured in DMEM (Invitrogen) with 10% foetal bovine serum (Invitrogen). Cells were transfected with the TurboFect transfection reagent (Fermentas) according to the manufacturer’s instructions. U2OS cells were transfected with GEMC1-HA and other constructs as indicated and were collected 24 h post-transfection. Immunoprecipitation of GEMC1-HA was performed using an anti-HA antibody (12CA5, Santa Cruz) as described in Pefani et al. (2011 ▸ ). Immunoprecipitates and total cell extracts corresponding to 10% of immunoprecipitates were analysed by Western blotting using anti-HA (Molecular Probes), anti-GFP and anti-Geminin (Xouri et al., 2004 ▸ ; Iliou et al., 2013 ▸ ) antibodies.
9
Monitoring NF-κB Activation with Dual-Luciferase Reporter Assay
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The assay was performed according to the instructions in the Dual-Luciferase Reporter kit (Promega). Briefly, HEK293T cells were grown overnight in DMED media to 70% confluence on top of poly-lysine (P8920, Sigma)-coated glass cover slips in 24-well plates, co-transfected with 0.2 μg and 0.025 μg of pNF-κB-luc and pRL-TK Renilla luciferase vector (Promega) DNA, respectively, using the TurboFect transfection reagent (R0531, Fermentas). After 24 hours, the medium was replaced with fresh DMEM and the cells were infected for 3 hours with bacteria from an overnight bacteria culture at a MOI of 1:100. The medium was then supplemented with gentamicin (100 μg/μl) in order to stop bacterial replication and avoid significant cell death. Cells were incubated for another 3 hours to allow accumulation of the reporter gene expression to detectable levels. Relative luminescence units (RLU) were normalized to those of uninfected cells. All experiments were repeated at least three times and significance was tested using the student T-test (un-paired, two-tailed).
10
Cloning and Mutagenesis of STAT2 and IRF9 Plasmids
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Human STAT2-3xHA-Migr1, mouse STAT2-Migr1, human IRF9-Migr1 and mouse ΔmSTAT2-3xHA-Migr1 plasmids were constructed in the following way: the full-length cDNA sequence of IRF9 was cloned into the XhoI and EcoRI restriction sites of the MigR1 plasmid [22 (link)]. The STAT2 and STAT2-ΔTAD coding sequences (2769 bp and 2199 bp, respectively) combined with the human influenza virus haemagglutinin (HA) epitope (3xHA, 116 bp) were sequentially cloned into the BglII and EcoRI restriction sites of Migr1. The STAT2-Y690F plasmid was constructed using the QuikChange site-directed mutagenesis kit (Agilent). Human STAT2-3xHA-Migr1 plasmid was used as a template and the following primers were designed to introduce the point mutation: For_hSTAT2_Y690F: CAGGAACGGAGGAAATTCCTGAAA-CACAGGCTC; Rev_hSTAT2_Y690F: GAGCCTGTGTTTCAG-GAATTTCCTCCGTTCCTG.
Two transfection methods were used: calcium phosphate method was used as described before [20 (link)], and TurboFect transfection reagent was used according to the manufacturer's descriptions (Fermentas).
Two transfection methods were used: calcium phosphate method was used as described before [20 (link)], and TurboFect transfection reagent was used according to the manufacturer's descriptions (Fermentas).
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