Turbofectin 8

Manufactured by OriGene

Sourced in United States

TurboFectin 8.0 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, including plasmid DNA, siRNA, and mRNA, into a wide range of mammalian cell lines. The product is optimized for high transfection efficiency while maintaining low cytotoxicity.

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TurboFectin (30 citations) TurboFectin 8.0 transfection reagent (15 citations) TurboFectin 8.0 reagent (11 citations)

60 protocols using turbofectin 8

Inducible Gene Expression in Cybrids

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Cybrids were plated in 60-mm dishes, incubated in glucose medium for 24 h, and then transfected with 2 μg of the full or empty vector by using Turbofectin 8 (Origene Technologies, Inc., Rockville, MD, USA) according to the manufacturer's protocol.
Six hours after transfection, 2 mM tetracycline (Sigma, St Louis, MO, USA) was added to the medium of pcDNA5/FRT/TO transformants while 150 μM isopropyl-b-D-1-thiogalactopyranoside (IPTG, Sigma) was added to the medium of pTUNE transformants for vector induction. All the media used for the successive experiments were supplemented with constant concentrations of these inducers.

Perli E., Giordano C., Pisano A., Montanari A., Campese A.F., Reyes A., Ghezzi D., Nasca A., Tuppen H.A., Orlandi M., Di Micco P., Poser E., Taylor R.W., Colotti G., Francisci S., Morea V., Frontali L., Zeviani M, & d'Amati G. (2014). The isolated carboxy-terminal domain of human mitochondrial leucyl-tRNA synthetase rescues the pathological phenotype of mitochondrial tRNA mutations in human cells. EMBO Molecular Medicine, 6(2), 169-182.

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Urchin Egg Ca2+ Signaling Assays

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Sea urchin egg homogenates were prepared and loaded with Ca²⁺ as described previously (56 (link)). Ca²⁺ release was measured by cuvette-based fluorimetry using the Ca²⁺ indicators fluo-3 or fluo-4 (3 μM, Invitrogen). SKBR3 cells were cultured as described previously (16 (link)) and transfected with TurboFectin 8 (Origene) using a 3:1 ratio of reagent:plasmid. The C-terminally GFP-tagged TPC1 construct from Stronglylcentrotus purpuratus was described in (44 (link)). Ca²⁺ imaging using Fura-2 and NAADP microinjection (pipette concentration 1 μM) were performed as described in (23 (link)). Fluorescence intensity (Fluo dyes) and ratio (Fura-2) values were normalized to values prior to stimulation and presented as F/F₀ or R/R₀, respectively. All agonists and drugs were from Sigma.

Rahman T., Cai X., Brailoiu G.C., Abood M.E., Brailoiu E, & Patel S. (2014). Two-pore channels provide insight into the evolution of voltage-gated Ca2+ and Na+ channels. Science signaling, 7(352), ra109.

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Investigating PPARγ Overexpression Effects on TBBPA

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To determine whether overexpression of PPARγ modified the effects of TBBPA (a potent PPARγ ligand based on ToxCast data), HepG2 cells were plated and transfected with either a negative control (NC) cloning plasmid (pCMV6-XL4, OriGene, Rockville, MD, USA) or an expression plasmid containing human untagged PPARγ clone (SC124177, OriGene, Rockville, MD, USA) following the manufacturer’s protocol. Briefly, 100 ng/well of either NC plasmid or PPARγ expression plasmid were diluted in Opti-MEM reduced serum media (Thermo Fisher Scientific, Waltman, MA, USA). TurboFectin 8.0 (OriGene, Rockville, MD USA) (0.3 µL/well) was added to plasmids diluted in Opti-MEM and incubated for 15 min at room temperature to form complexes. Following incubation, plasmid:TurboFectin 8.0 complexes were added to each well and gently shaken to distribute evenly. Transfected HepG2 cells were then allowed to incubate for 48 h followed by exposure to either vehicle (0.1% DMSO) or 30 µM TBBPA for 24 h as described above. PPARγ protein levels in situ across transfections and treatments were then quantified using a 1:150 dilution of a human PPARγ-specific antibody (E-8, sc-7273; Santa Cruz Biotechnology, Dallas, TX, USA) following previously described protocols (Cheng et al., 2021 (link)). Transfected cells were also collected for cell viability, ORO neutral lipid staining, and FASN IHC as described above.

Cheng V, & Volz D.C. (2022). Halogenated bisphenol a analogues induce PPARγ-independent toxicity within human hepatocellular carcinoma cells. Current Research in Toxicology, 3, 100079.

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SIRT5 Overexpression and Silencing in Cells

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MDA-MB-231 and C2C12 cells were stably transfected with a pcDNA3.1 expression vector encoding human and mouse SIRT5-Flag (Addgene, 13816) for overexpression and for human SIRT5 and mouse Sirt5 shRNAs for silencing. Stable clones were generated by delivering plasmid DNA constructs into cells using TurboFectin 8.0 (Origene Technologies, TF81001) according to the manufacturer's recommendations. Briefly, cells were seeded on a 24-well plate. The following day the cells were transfected. TurboFectin reagents were first mixed with serum-free RPMI at room temperature for 5 min. Subsequently, plasmid DNA was added to the TurboFectin-containing media and incubated at room temperature for 30 min. After that, the mixtures were added to the cells. The selection of stable overexpressing and silenced clones was started 24 h later with the addition of 500 μg/ml of geneticin (Sigma-Aldrich, A1720).

Polletta L., Vernucci E., Carnevale I., Arcangeli T., Rotili D., Palmerio S., Steegborn C., Nowak T., Schutkowski M., Pellegrini L., Sansone L., Villanova L., Runci A., Pucci B., Morgante E., Fini M., Mai A., Russo M.A, & Tafani M. (2015). SIRT5 regulation of ammonia-induced autophagy and mitophagy. Autophagy, 11(2), 253-270.

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Plasmid and Lentiviral Transfection Protocols

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Plasmid transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific) or Turbofectin 8.0 (OriGene) according to suppliers’ instructions. Lentiviruses were produced by transfecting 293T cells with a lentiviral and two packaging plasmids (psPax2 and pMD2.G). Culture media containing lentiviral particles were harvested after 48 h and used to infect cells in the presence of 4 µg ml⁻¹ polybrane. After selection with 2 µg ml⁻¹ puromycin for 48 h, cells were cultured in the absence of puromycin for at least 24 h. siRNA transfection was performed using RNAiMAX (Thermo Fisher Scientific). The target sequences of siRNAs are as follows: siControl, 5′-CGUACGCGGAAUACUUCGA-3′; siFAM111A.271, 5′-CUAAAGAGCAACAGAAUAA-3′; siFAM111A.1213, 5′-CGAUUAAAGUAGUGAAACU-3′. siRNA oligo sequences are shown in Supplementary Table 3.

Kojima Y., Machida Y., Palani S., Caulfield T.R., Radisky E.S., Kaufmann S.H, & Machida Y.J. (2020). FAM111A protects replication forks from protein obstacles via its trypsin-like domain. Nature Communications, 11, 1318.

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Regulation of PMCs by miR-17-92 and E2F1

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PMCs were transfected with mixed miRNA mimics of miR-17-92 components, mixed miRNA inhibitors of miR-17-92 components, or scrambled miRNAs (Ribobio, Guangzhou, China) at a concentration of 50nM using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacture’s protocol. Total RNA and protein were isolated from the transfected cells, followed by Real-time PCR and Western blot.
Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol. After 6 h of transfection, the medium was replaced by serum-containing medium.

Li L., Shi B., Chen J., Li C., Wang S., Wang Z, & Zhu G. (2017). An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells. Scientific Reports, 7, 5148.

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CRISPR-mediated protein tagging and degradation

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pX330A_sgX_sgPITCh cutting plasmid and pCRIS-PITChv2 repair template plasmids (provided by Winter laboratory, partly available via Addgene 91793, 91796) were cloned for each target gene according to Brand et al.^{49 (link)} and transfected into HAP1 cells using TurboFectin 8.0 (OriGene). After puromycin selection, single-cell clones were grown up and validated for proper insertion and function of the tag by PCR, Sanger sequencing and western blot analyses. dTAG7, dTAG13 or dTAG47 were tested for their degradation dynamics and then dTAG47 was used for all experiments to induce the degradation of the tagged proteins^{40 (link),50 (link),51 (link)}.

Schick S., Grosche S., Kohl K.E., Drpic D., Jaeger M.G., Marella N.C., Imrichova H., Lin J.M., Hofstätter G., Schuster M., Rendeiro A.F., Koren A., Petronczki M., Bock C., Müller A.C., Winter G.E, & Kubicek S. (2021). Acute BAF perturbation causes immediate changes in chromatin accessibility. Nature genetics, 53(3), 269-278.

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Transfection and Live-Cell Imaging of Cell Lines

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Human HeLa cells were obtained from Dr David Murray (University of Alberta) and cultured in Dulbecco's modified Eagle's medium (DMEM)-F12 media supplemented with 10% fetal calf serum (FCS). Chinese Hamster Ovary EM9 cells were kindly provided by Dr Keith Caldecott (University of Sussex) and cultured in DMEM supplemented with 10% FCS. PARP1^+/+ (F20) and PARP1^−/− (A1) mouse embryo fibroblasts (MEFs) were kindly provided by Dr Zhao-Qi Wang (Jena University, Germany) and cultured in DMEM low glucose media supplemented with 10% FCS. For PARP-1^−/− MEFs, growth media contained neomycin at a final concentration of 600 μg/ml. For transfection, cells were plated in 35-mm glass bottom dishes (MatTek Corporation, Ashland, MA, USA) and allowed to attach over 24 h. Then, cells were transfected with DNA constructs of interest using Turbofectin 8.0 (OriGene, Rockville, MD, USA) according to the manufacturer's protocol. Cells were used for live cell imaging 24–48 h post-transfection.

Abdou I., Poirier G.G., Hendzel M.J, & Weinfeld M. (2014). DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair. Nucleic Acids Research, 43(2), 875-892.

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CRISPR/Cas9-Mediated Knockout of IFITM1 in Breast Cancer Cells

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SUM149 and MDA-MB-157 cells were subjected to CRISPR/Cas9-mediated
knockout of IFITM1 by lentiviral transduction using particles from
OriGene™ Technologies (Catalog number: KN201617). The guide RNA vector
5’- TGATCACGGTGGACCTTGGA-3’ or a scrambled control was cloned into
a pCas-Guide vector which expresses Cas9 behind CMV and U6 promoters. This
vector was co-transfected with the donor template including homologous arms and
a functional GFP-puromycin cassette using Turbofectin 8.0 (Origene: #TF81001) as
the delivery reagent. For SUM149, cells were passaged at a 1:10 ratio 48 hours
post-transfection for 8 passages. Cells were treated with 1000μg/mL
puromycin daily. Single cells were grown in puromycin until colonies formed.
Both clonal populations and a pooled population of all puromycin resistant cells
were expanded and screened for absence of IFITM1 protein expression using the
IFITM1 antibody. The pooled population of CAS9/Control cells and IFITM1 KO cells
were then used for functional in vitro assays as described
below in section 2.8. For MDA-MB-157,
transfected cells were passaged at a 1:2 ratio for 8 passages prior to treatment
with 250–350μg/mL puromycin for one week.

Provance O.K., Geanes E.S., Lui A.J., Roy A., Holloran S., Gunewardena S., Hagan C., Weir S, & Lewis-Wambi J. (2021). Disrupting interferon-alpha and NF-kappaB crosstalk suppresses IFITM1 expression attenuating triple-negative breast cancer progression. Cancer letters, 514, 12-29.

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Retroviral Transduction of 3T3-L1 Cells

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One day prior to transfection, Phoenix Eco cells (NGVB, Indianapolis) were seeded at a density of 3 × 10⁶/10 cm dish. Retroviral vector was transfected into cells using TurboFectin 8.0 (Origene) according to the manufacturer’s instructions. Two days later, the medium containing viruses was filtered through 0.45 μm syringe filter and was used to infect 3T3-L1 cells. Two days after infection, cells were selected with 2.5 μg/ml puromycin or 6 ug/ml Blasticidine. For PPARγ2 overexpression experiment: pMSCVpuro- PPARγ2 was obtained from Addgene and control vector pMSCVpuro from Clontech. For DPP8 or DPP9 knockdown: DPP8 shRNA plasmid (Origene, TF504979) was puromycin resistance, with sequence 5′ TTCCTGAGTCTGGAGAACACTATGAACTG-3′ ; DPP9 shRNA plasmid (Origene, TG515456) was puromycin resistance, with sequence 5′- TGTCAAGCTGCGAGAAGGAACTGGTACAG-3′. For DPP8 and DPP9 double-knockdown: DPP9 shRNA sequence was constructed into blasticidine resistance vector pGFP-B-RS and co-transfected with puromycin resistance DPP9 shRNA plasmid.

Han R., Wang X., Bachovchin W., Zukowska Z, & Osborn J.W. (2015). Inhibition of dipeptidyl peptidase 8/9 impairs preadipocyte differentiation. Scientific Reports, 5, 12348.

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