Fugene hd transfection reagent

Manufactured by Promega

Sourced in United States, Germany, United Kingdom, Japan, China, France, Canada, Australia, Italy, Switzerland

FuGENE HD is a transfection reagent that facilitates the delivery of nucleic acids, such as plasmid DNA, into mammalian cells. It is designed to enhance transfection efficiency in a variety of cell lines.

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FuGENE HD DNA transfection reagent (4 citations) FuGENE HD Transfection Reagent Kit (2 citations) FuGENE® HD Transfection Reagent-to-DNA ratio (2 citations) FuGENE HD in vitro Transfection Reagent (2 citations) FuGENE HD transfection (14 citations) Fugene HD reagent (207 citations) FuGENE transfection reagent (195 citations) HD transfection reagent (70 citations) FuGENE HD (2156 citations) FuGENE reagent (62 citations)

1 708 protocols using fugene hd transfection reagent

Luciferase Reporter Assay Protocol

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Transfection of cells was performed by using the FuGENE ® HD transfection reagent (Promega, Mannheim, Germany) according to the manufacturer's instructions.
For the luciferase reporter assay, HCT116 cells were seeded into a 24-well plate (62,500 cells per well) in a total volume of 0.5 ml/well of cell culture medium and cultured overnight. Cells were then transfected with FuGENE ® HD transfection reagent using a total of 0.5 g of plasmid DNA (transfection mixture per well: 0.05 g of promoter reporter plasmid, 0.45 g of expression plasmid(s), 1.25 l FuGENE ® HD transfection reagent and 25 l cell culture medium without FCS).
To measure luciferase activity, cells were collected at given time points after transfection by lysing in cell culture lysis reagent (Promega) and measured with the Luciferase Assay System (Promega) following the manufacturer's recommendations.
For Western blot analysis, cell lysates were centrifuged at 13,000 × g to remove cell debris. The protein content was determined with the Bradford method using the BioRad protein assay reagent (BioRad, Munich, Germany). Protein extracts were immediately used for Western blot analysis or stored at -20 • C.

Schwind L., Zimmer A.D., Götz C, & Montenarh M. (2015). CK2 phosphorylation of C/EBPδ regulates its transcription factor activity. The international journal of biochemistry & cell biology, 61.

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Effective siRNA-mediated KLF2 Knockdown

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Three different siRNA duplexes used for KLF2 knockdown (Table 2) were designed and synthesized by Genepharma company (Shanghai, China). The 152 pmol given siRNA duplexes or the mixtures of 152 pmol given siRNA duplexes and 2 μg pCMV-myc-KLF2 were transfected into the chicken preadipocytes cultured in one well of the 6-well culture plate using FuGENE HD transfection reagent (Promega). After transfection for 48 h, semi-quantitative RT-PCR and western blot was employed to detect the knockdown efficiency of these siRNA duplexes. Only the siRNA duplexes with the highest knockdown efficiency were used for subsequent studies. Briefly, an amount of 152 pmol given siRNA duplexes was transfected into the chicken preadipocytes cultured in each well of 6-well culture plate using FuGENE HD transfection reagent (Promega). After transfection for 48 h, western blot and real-time RT-PCR were recruited to study the effect of KLF2 knockdown on C/EBPZ expression.Table 2

The siRNA sequences used for KLF2 knockdown.

Table 2

Name	Sense (5′→3′)	Antisense (5′→3′)
siRNA-KLF2-1	GAGAAAGCGCUCCACGAAATT	UUUCGUGGAGCGCUUUCUCTT
siRNA-KLF2-2	GGAGGCUUCUACCAGACAATT	UUGUCUGGUAGAAGCCUCCTT
siRNA-KLF2-3	GCCCUGAGAUGGACUCCAATT	UUGGAGUCCAUCUCAGGGCTT
siRNA-NC	CCAAGAGCAGCUCCUUUAATT	UUAAAGGAGCUGCUCUUGGTT

Gao L., Wang Y., Gao Q., Chen Y, & Zhang Z. (2024). Transcriptional control of CCAAT/enhancer binding protein zeta gene in chicken adipose tissue. Poultry Science, 103(4), 103540.

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Promoter and sgRNA Activity Assays

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One day before transfection, BFFs were seeded at a density of 1 × 10⁵ cells per well of a 24-well plate for assaying the promoter of bROSA26 gene. Approximately 0.5 μg of plasmids (0.4 μg for pGL4.10-promoter or empty vector pGL4.10 and 0.1 μg for pRL-TK) was cotransfected according to the protocol of FuGENE HD Transfection Reagent (Promega). The pRL-TK plasmid vector was used as an internal reference vector for standardizing transfection efficiency.
One day before transfection, 293T cells were seeded at a density of 2 × 10⁵ cells per well of a 24-well plate for assaying the activity of sgRNAs. Approximately 0.8 μg of plasmids (0.18 μg for SSA reporter plasmid and 0.6 μg Cas9 expression plasmids containing 20-nt guide sequence or pSpCas9(BB)-2A-Puro and 0.02 μg for pRL-SV40) was cotransfected according to the protocol of FuGENE HD Transfection Reagent (Promega). The pRL-SV40 plasmid vector was used as an internal reference vector for standardizing transfection efficiency.
Cell lysates were collected 48 h posttransfection and prepared for luciferase activity analysis using the Double-Luciferase Reporter Assay Kit (TransGen Biotec) following the manufacturer's instructions. Relative luciferase activities were expressed as the ration of the luciferase value to the Renilla value.

Yuan M., Zhang J., Gao Y., Yuan Z., Zhu Z., Wei Y., Wu T., Han J, & Zhang Y. (2021). HMEJ-based safe-harbor genome editing enables efficient generation of cattle with increased resistance to tuberculosis. The Journal of Biological Chemistry, 296, 100497.

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Transient Transfection Assay for AhR and CD274 Activity

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HEK293 cells were used in the transient transfection system, as previously described⁶⁴. In brief, 20,000 cells per well were plated in 96-well flat-bottom plates. For the assessment of AhR agonistic activity, cells were transfected with pGud-Luc21 and pTK-Renilla (Renilla luciferase under control of constitutively active thymidine kinase promoter, Promega, Madison, WI) using Fugene-HD Transfection Reagent (Promega, #E2311) as suggested by the manufacturer 24 h after plating. After 24 h, transfected cells were incubated with DMEM supplemented with 10% of patient serum in duplicates. Luciferase activity was analyzed 24 hours later using the Dual Luciferase Reporter System (Promega, #E1980). Firefly luciferase activity was normalized to Luciferase activity to determine relative AhR activity. Results are represented as percent normalized AhR activity relative to control samples.
For the assessment of CD274 promoter activity, cells were transfected with pEZX-PF02 (eGFP under control of CD274 promoter) using Fugene-HD Transfection Reagent (Promega, #E2311) as suggested by the manufacturer. After 24 h, transfected cells were stimulated with the indicated stimuli. After 24 h stimulation, cells were harvested and the promoter activation indicated by the GFP signal was analyzed by conventional flow cytometry.

Linnerbauer M., Beyer T., Nirschl L., Farrenkopf D., Lößlein L., Vandrey O., Peter A., Tsaktanis T., Kebir H., Laplaud D., Oellinger R., Engleitner T., Alvarez J.I., Rad R., Korn T., Hemmer B., Quintana F.J, & Rothhammer V. (2023). PD-L1 positive astrocytes attenuate inflammatory functions of PD-1 positive microglia in models of autoimmune neuroinflammation. Nature Communications, 14, 5555.

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Innate Immune Signaling Pathway Analysis

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HeLa cells, HEK293 cells, MARC-145 cells and PAMs were plated in 6-well culture plates at 70–80% confluence and transfected with poly (I:C), 5′-ppp dsRNA, ssRNA DR, ssRNA 41 at a concentration of 2 μg/mL or mock transfected by HiPerFect Transfection Reagent (Qiagen) for 24 h. The cell lysates were harvested and subjected to real-time RT-PCR and western blot analysis.
For shRNA transfection, MARC-145 cells and HEK293 cells were plated in 6-well culture plates at 70–80% confluence and transfected with 4 μg of shRNA targeting human STAT1 or shRNA control by FuGENE^® HD transfection reagent (Promega) for 48 h. Then the cells were selected using medium containing 50–150 μg/mL Zeocin (Life technologies) for 3 days until cell foci were identified. The selected cells were used for further study.
For luciferase reporter assay, the indicated plasmids were transfected into 5 × 10⁴ HeLa cells in 24-well culture plates along with pRL-TK as an internal reference control, using the FuGENE^® HD transfection reagent (Promega) according to the manufacture’s guidelines. After 24 h transfection, the cells were harvested and subjected to luciferase assay.

Yang S., Zhan Y., Zhou Y., Jiang Y., Zheng X., Yu L., Tong W., Gao F., Li L., Huang Q., Ma Z, & Tong G. (2016). Interferon regulatory factor 3 is a key regulation factor for inducing the expression of SAMHD1 in antiviral innate immunity. Scientific Reports, 6, 29665.

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Validated PIM3 Expression Vector Construction and Transfection

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The PIM3 expression vector, pcDNA3.1/V5-His-PIM3, was a generous gift from Dr. Jussi Taipale and was generated by PCR amplification and cloning into the pcDNA3.1/V5-HisC vector (19 (link)). The plasmid was sequenced for verification (Genomics Core, UAB). Empty vector (EV, pcDNA3.1/V5-HisC) was used as a control. Transfection was carried out using FuGENE^® HD Transfection Reagent (Promega) per the manufacturer’s protocol. Briefly, HuH6 PIM3 KO cells were plated on the day prior to transfection. The appropriate plasmid was incubated for 15 minutes at room temperature in Opti-MEM^™ media (Thermo Fisher Scientific) with FuGENE^® HD Transfection Reagent (Promega) in a 3:2 ratio of transfection reagent to DNA with 7.5 μg DNA per 1 × 10⁶ cells and added to the cells while swirling the flask. Cells were transfected with either pcDNA3.1/V5-HisC (EV) or pcDNA3.1/V5-HisC-PIM3 (PIM3 expression vector) for 48–72 hours prior to use in experiments. Lysates were made to perform immunoblotting, as described above, to assess for PIM3 expression.

Marayati R., Stafman L.L., Williams A.P., Bownes L.V., Quinn C.H., Markert H.R., Easlick J.L., Stewart J.E., Crossman D.K., Mroczek-Musulman E, & Beierle E.A. (2021). CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells. Cancer gene therapy, 29(5), 558-572.

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Validated PIM3 Expression Vector Construction and Transfection

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Overexpression of MBLAC1 in HeLa and HEK293 Cells

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HeLa (ATCC) and HEK293 (kindly provided by Prof. Peter McHugh) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, and 2 mM L-glutamine, at 37°C in a humidified incubator (5% CO₂). To generate the construct MBLAC1 6xHis-pCDNA 3.1, an appropriate sequence coding for MBLAC1 (GeneART, Thermo Fisher Scientific) was amplified by PCR to introduce a C-terminal 6xHis tag, then cloned into the pCDNA 3.1 vector (Invitrogen). Both HeLa and HEK293 cells were transiently transfected with the MBLAC1 6xHis-pCDNA3.1 construct using the Fugene HD transfection reagent (Promega), following the manufacturer’s instructions. MBLAC1 production was monitored by western blotting, 24 and 48 hr after transfection. To generate a stable cell line overexpressing MBLAC1, HEK293 cells were transfected with the MBLAC1 6xHis-pCDNA 3.1 construct using the Fugene HD transfection reagent (Promega), following the manufacturer’s instructions. Stably transfected cells were selected by addition of geneticin (G418) (1 mg/ml) to the cell culture media; cell clones were generated by limiting dilution plating. Clones were analysed by western blotting for their capacity to produce MBLAC1.

Pettinati I., Grzechnik P., Ribeiro de Almeida C., Brem J., McDonough M.A., Dhir S., Proudfoot N.J, & Schofield C.J. (2018). Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease. eLife, 7, e39865.

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Plasmid and siRNA Transfection in Cell Lines

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For plasmid DNA, HeLa cells were transfected with 500 ng of wild type (WT) pGL3-Basic-ADTRPp-Luc or mutant reporters together with 25 ng of Renilla luciferase reporter plasmid using Lipofectamine^®2000 (Invitrogen, Thermo Fisher Scientific, the Massachusetts, USA) according to the manufacturer's instruction. Transfection of EAhy926 cells was carried out using the Fu GENE^® HD Transfection Reagent from Promega (Madison, Wisconsin, USA).
Transfection of siRNA was performed for HeLa and EAhy926 cells using Lipofectamine^® RNAi MAX (Invitrogen, Thermo Fisher Scientific, USA) and Fu GENE^®HD Transfection Reagent (Promega, Madison, Wisconsin, the USA), respectively.

Luo C., Pook E., Tang B., Zhang W., Li S., Leineweber K., Cheung S.H., Chen Q., Bechem M., Hu J.S., Laux V, & Wang Q.K. (2017). Androgen inhibits key atherosclerotic processes by directly activating ADTRP transcription. Biochimica et biophysica acta, 1863(9), 2319-2332.

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Generation of FUS knockout and overexpression SH-SY5Y cell lines

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The FUS knockout (FUS^KO) stable SH-sy5y cell line was constructed by FUS/TLS CRISPR KO Plasmids (Cat No. Sc-400612-NIC-2, Santa Cruz). Cells were transfected plasmids once they reached 70–80% confluency using FuGENE HD transfection reagent (Cat No. E2311, Promega) at a 3:2 FuGENE® HD Transfection Reagent: DNA ratio. After 48 h incubation, successful transfection of CRISPR/Cas9 KO Plasmid was visually confirmed by cell fluorescence. The lowest concentration of Puromycin that killed 100% of non-transfected cells in 3 days from the start of Puromycin selection was determined. Finally, the cells were selected with Puromycin (Cat No. 60209ES10, Yeasen) at concentration of 2.5 μg/ml for 7 days, followed by Puromycin maintenance.
pLV [Exp]-mCherry/Neo-EF1A > FLAG/hFUS [NM_004960.4] vectors (VectorBuilder) was transfected to FUS^KO cell line to generate co-transfection of FUS knockout and FUS overexpression (FUS^KO + OE) SH-sy5y cell line. Finally, the cells were selected with G418 (Cat No. 108321-42-2, Aladdin) at a concentration of 600 μg/ml for 14 days, followed by G418 maintenance. The successful construction of FUS^KO + OE and FUS^KO stable SH-sy5y strains was confirmed by western blotting.

Wu Y., Huang X., Tan Z., Zang J., Peng M., He N., Zhang T., Mai H., Xu A, & Lu D. (2023). FUS-mediated HypEVs: Neuroprotective effects against ischemic stroke. Bioactive Materials, 29, 196-213.

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