Superfect transfection reagent

Manufactured by Qiagen

Sourced in Germany, United States, United Kingdom

SuperFect transfection reagent is a polycationic dendrimer-based reagent designed to facilitate the delivery of DNA, RNA, or other macromolecules into a variety of mammalian cell types. It functions by forming complexes with the biological cargo, which are then taken up by the target cells through endocytosis.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (13 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions) Dual luciferase assay system, by Promega (5 mentions) Fetal bovine serum (fbs), by Merck Group (5 mentions) Pegfp f, by Takara Bio (5 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Streptomycin, by Thermo Fisher Scientific (4 mentions) Luciferase assay system, by Promega (4 mentions) Penicillin, by Thermo Fisher Scientific (4 mentions) Phrg tk vector, by Promega (3 mentions) Horse serum, by Thermo Fisher Scientific (3 mentions) Pdsred monomer f, by Takara Bio (3 mentions) Hiperfect transfection reagent, by Qiagen (3 mentions) Rnaimax, by Thermo Fisher Scientific (3 mentions) Pgl3 basic vector, by Promega (2 mentions) Sv 40 renilla luc, by Promega (2 mentions) Dual luciferase reporter assay kit, by Promega (2 mentions) Prl sv40 vector, by Promega (2 mentions) Hek293t, by American Type Culture Collection (2 mentions)

Spelling variants of this product

Superfect (166 citations) Superfect reagent (63 citations) Transfection reagent (24 citations) SuperFect Transfection (4 citations)

177 protocols using superfect transfection reagent

Transcriptional Regulation of FTO by PU.1 and m6A Modulation

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The DNA fragments of different lengths of the FTO promoter were amplified from human genomic cDNA by PCR using the primers listed in Additional file 2: Table S3 and inserted into the pGL3-basic vector (Promega) between KpnI and HindIII enzyme sites. The pcDNA3.1 vector containing DNA fragments of human PU.1-CDS were synthesized by Vigene Biosciences, Inc. Total 400 ng of pcDNA3.1 vector (i.e., pcDNA3.1-mock or pcDNA3.1-PU.1-CDS) and pGL3-basic vector (i.e., PGL3-P1 to PGL3-P4), and 20 ng pRL-TK Renilla luciferase reporter vector were cotransfected into HEK293T cells in 24-well plates using the SuperFect® Transfection Reagent (Qiagen). Luciferase activity was assessed 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega). Each experiment was repeated in triplicate.
The wild-type and m⁶A-mutant IGFBP2 3′ untranslated region (3′-UTR) were directly synthesized and inserted into the psiCHECK2 vector (Promega) by Vigene Biosciences, Inc. Briefly, 400 ng wild-type or mutant psiCHECK2-IGFBP2-3′-UTR and 100 ng FTO-expressing vector (i.e., pCMV6-wtFTO, pCMV6-mutFTO, or pCMV6-mock) were cotransfected into HEK-293T cells in 24-well plates with the SuperFect® Transfection Reagent (Qiagen). Relative luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega) 48 h posttransfection. Each experiment was repeated in triplicate.

Zhou W., Li S., Wang H., Zhou J., Li S., Chen G., Guan W., Fu X., Nervi C., Yu L, & Li Y. (2024). A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia. Experimental Hematology & Oncology, 13, 9.

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Investigating TLR-mediated EV71 Infection

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Endotoxin-free TLR-related and EV71 capsid recombinant eukaryotic plasmids were prepared using the E.Z.N.A. Endo-free Plasmid Kit (OMEGA, USA), according to the manufacturer’s instructions. HEK293 and RD cells were seeded in a 6-well plate and the TLR-related or EV71 capsid recombinant eukaryotic plasmids were transiently single or co-transfected into host cells at the indicated dose (1 or 2 μg) using the SuperFect Transfection Reagent (Qiagen, Germany), according to the manufacturer’s instructions.
After transfecting the plasmids into cells for 24 h, TLR-overexpressing HEK293 and RD cells were infected with EV71 at a multiplicity of infection (MOI) of 0.5 or 1. Then, cytopathic effects (CPEs) on non-transfected and transfected HEK293 and RD cells were observed 24 h after EV71 infection using a microscope (Olympus, Japan).

Sun P.P., Li D., Su M., Ren Q., Guo W.P., Wang J.L., Du L.Y, & Xie G.C. (2023). Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response. Frontiers in Immunology, 14, 1187035.

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Dual-Luciferase Assay for NF-κB Activity

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Cells cultured in six-well plates were transfected with an empty vector, lin28b-P1-Luc containing an NF-κB binding site (GGGGCTTTC) in the first intron, or a mutant lin28b-P1(GGGGCTTTC→TCATACGAT)-Luc (2 (link), 5 (link)) and, as an internal control, SV-40-Renilla-Luc (Promega) in the presence of Superfect transfection reagent (Qiagen). After 48 h, the transfected cells were left untreated or treated with the indicated agents, lysed with passive lysis buffer and then the lysates were analyzed using the dual luciferase assay system (Promega).

Alam M., Ahmad R., Rajabi H, & Kufe D. (2014). MUC1-C INDUCES THE LIN28B→LET-7→HMGA2 AXIS TO REGULATE SELF-RENEWAL IN NSCLC. Molecular cancer research : MCR, 13(3), 449-460.

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Chromatin Immunoprecipitation of Sp1 and Sp3 Transcription Factors

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Using Superfect transfection reagent (Qiagen), HPAEC (approximately 2×10⁶ cells per dish, six dishes per transfection) were either mock transfected or transfected with 5 μg of pCP2-Tat₁₀₁ plasmid. Twenty-four hours after transfection, cells were cross-linked with 1% formaldehyde and quenched with 1X Glycine followed by two consecutive washes with ice-cold 1X PBS. Cells were collected via scraping in 1X PBS supplemented with protease and phosphatase inhibitors and centrifuged at 3000 × g for 5 minutes. Following the manufacturer’s instructions, the Pierce Agarose ChIP Kit (Thermo Scientific) was used to precipitate and clean-up DNA bound by either control IgG (3 μg rabbit or mouse IgG provided in the kit), anti-Sp1 rabbit polyclonal (3 μg, ChIP-grade, Abcam) or Sp3 mouse monoclonal (3 μg, ChIP-grade, Santa Cruz). The purified final DNA was quantified using Qubit® 2.0 Fluorometry (Invitrogen Life Technologies).

Manes T.L., Simenauer A., Geohring J.L., Flemming J., Brehm M, & Cota-Gomez A. (2019). The HIV-Tat protein interacts with Sp3 transcription factor and inhibits its binding to a distal site of the sod2 promoter in human pulmonary artery endothelial cells. Free radical biology & medicine, 147, 102-113.

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Measuring SOD2 Transcriptional Regulation

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To measure sod2 transcription we used promoter-reporter plasmids expressing luciferase under the control of increasingly longer sod2 upstream sequences (Fig.1) in HPAEC, HeLa WT and HeLa-Tat_III cells. Cells were transiently transfected using Superfect transfection reagent according to the manufacturer’s protocol (Qiagen). HeLa WT and HeLa-Tat_III cells were transfected with 1 μg of total DNA, 0.5 μg of sod2 promoter-reporter plasmid and 0.5 μg of a CMV driven Renilla luciferase plasmid. HPAEC were also co-transfected with 0.5 μg of the Tat-expressing plasmid, pCP2-Tat₁₀₁ (see Supplemental Materials Table 2). Twenty-four hours after transfection, luciferase was measured using the Dual Luciferase Reporter Assay System (Promega,) according to the manufacturer’s instructions in a dual injector MicroPlate Luminometer (Promega). All luminescence data was normalized to the Renilla signal.

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PPAR Luciferase Assay in Murine Mast Cells

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P815 murine mast cells were transfected with PPAR luciferase reporter using SuperFect transfection reagent (Qiagen) in Opti MEM® medium. PPAR reporter used for transfection was a mixture of a PPAR-responsive (inducible) Firefly luciferase construct and a constitutively expressing Renilla luciferase construct. A constitutively expressing GFP construct was used initially to optimize transfection conditions for P815 cells (~62%). After 24 hours of transfection, cells were treated with vehicle (control), CBD or a positive control PPARγ agonist, troglitazone. For in vitro assays the vehicle for CBD contained <0.1% DMSO at final concentrations. Luciferase assay was performed 24 hours following treatment using a commercially available Dual-Glo® luciferase assay system (Promega) and measured in a luminometer (Perkin Elmer). Values were expressed as normalized relative luciferase units and fold induction compared to control was calculated.

Hegde V.L., Singh U.P., Nagarkatti P.S, & Nagarkatti M. (2015). Critical role of mast cells and peroxisome proliferator-activated receptor gamma (PPARγ) in the induction of myeloid-derived suppressor cells by marijuana cannabidiol in vivo. Journal of immunology (Baltimore, Md. : 1950), 194(11), 5211-5222.

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Rescue and Propagation of Recombinant RABV

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Recombinant RABVs were rescued as described previously [55 ] and propagated in BSR cells. In brief, BSR cells were transfected with 2.0 μg of full infectious clone, 0.5 μg of pH-N, 0.25 μg of pH-P, 0.15 μg of pH-G, 0.1 μg of pH-L using the SuperFect transfection reagent (Qiagen, Valencia, CA) in accordance with the manufacture's protocol. At four days post transfection, the culture supernatant was replaced with fresh medium and incubated for another three days, and then the culture medium was collected and subjected to detect the rescued rRABV using FITC-conjugated anti-RABV N antibodies (Abs).

Wang Z., Liang Q., Zhang Y., Yang J., Li M., Wang K., Cui M., Chen H., Fu Z.F., Zhao L, & Zhou M. (2017). An optimized HMGB1 expressed by recombinant rabies virus enhances immunogenicity through activation of dendritic cells in mice. Oncotarget, 8(48), 83539-83554.

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CD24 Promoter Cloning and Analysis

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Genomic DNA was isolated from a male C57BL/6N mouse liver using the Genomic DNA isolation kit (QIAGEN, Germantown, MD, USA). The CD24 promoter region from −688 to −1 from the TSS was amplified from genomic DNA with the GC-Rich PCR system (Roche, Basil, Switzerland) using the primers indicated in Table 1. The promoter was cloned into the HindIII and BglII sites of the pGL4.17 vector (Promega, Madison, USA). The deletion constructs −469 to −1, −357 to −1, and −168 to −1 were generated using the Erase-a-base kit (Promega) according to the manufacturer's instructions. All sequences were verified by sequencing at The Centre for Applied Genomics (Toronto, ON, Canada).
Control or RasV12 cells (3 × 10⁴ cells/well in 24-well-plates) were transfected with 1 μg of the pGL4.17 vector with or without the CD24 promoter regions and 6.66 ng pRL-SV40 vector (Promega) using 2.5 μl Superfect transfection reagent (Qiagen), following the manufacturer's instructions. After 24 h, cells were lysed with 1X Passive Lysis Buffer and Firefly and Renilla Luciferase activity were measured using the Dual-Luciferase Reporter Assay kit (Promega).

Pallegar N.K., Ayre D.C, & Christian S.L. (2015). Repression of CD24 surface protein expression by oncogenic Ras is relieved by inhibition of Raf but not MEK or PI3K. Frontiers in Cell and Developmental Biology, 3, 47.

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Construction of CMV m74stop Mutant

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Markerless BAC mutagenesis was performed to introduce a stop cassette in the m74 ORF in the pSM3fr-MCK-2fl BAC as described previously [60 (link)]. For constructing the pSM3fr-m74stop mutant (virus: ΔgO; m74stop), the primers m74stop-for (5’-GGA GGT TCG GTC GCA TCG ATT GTA TCA TAA CCT CCG TCT TCA TAA TCA TCG GCT AGT TAA CTA GCC AGG ATG ACG ACG ATA AGT AGG G-3’) and m74stop-rev (5’-AAA GTG TAG CAT ACA ACC CGG CCG TTA CCG GCT ATA TCG AGA TGA GCG AAG GCT AGT TAA CTA GCC GAT GAT TAT GAA GAC GGA GGC AAC CAA TTA ACC AAT TCT GAT TAG-3’) were used. The sequences of the stop cassette are indicated by italic type. Insertion of the stop cassette was controlled by restriction enzyme pattern analysis and sequencing. Recombinant CMVs were reconstituted by transfection of purified BAC DNA into MEF using Superfect transfection reagent (Qiagen). Transfected cells were propagated until viral plaques appeared and supernatants from these cultures were used for further propagation. Virus stocks were prepared from supernatants of infected NIH3T3 cells, or from NIH-gO cells in case of gO-transcomplementation, by sucrose-gradient ultracentrifugation as described [41 ]. Virus titers were determined by TCID₅₀ assay or standard plaque assay performed on MEF.

Lemmermann N.A., Krmpotic A., Podlech J., Brizic I., Prager A., Adler H., Karbach A., Wu Y., Jonjic S., Reddehase M.J, & Adler B. (2015). Non-redundant and Redundant Roles of Cytomegalovirus gH/gL Complexes in Host Organ Entry and Intra-tissue Spread. PLoS Pathogens, 11(2), e1004640.

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Transfection and Cell Harvest Protocol

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293T cells were transfected with expression vectors using Superfect transfection reagent (Qiagen) following the manufacturer’s instructions. Typically, for a 6-well plate, 3 μg of DNA/well was transfected; and for a 10-cm plate, 20 μg of DNA were utilized. Forty-eight hours post-transfection, the cells were harvested and washed with PBS. Total RNA or total cell lysates were prepared and used for RT-qPCR or immunoblot analyses as indicated.

Geng C., Kaochar S., Li M., Rajapakshe K., Fiskus W., Dong J., Foley C., Dong B., Zhang L., Kwon O.J., Shah S.S., Bolaki M., Xin L., Ittmann M., O’Malley B.W., Coarfa C, & Mitsiades N. (2017). SPOP regulates prostate epithelial cell proliferation and promotes ubiquitination and turnover of cMYC oncoprotein. Oncogene, 36(33), 4767-4777.

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