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lipofectamine 3000  (Thermo Fisher)


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    Structured Review

    Thermo Fisher lipofectamine 3000
    Lipofectamine 3000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/lipofectamine+3000/Lapatinib/pmc13273694-118-18-20
    Average 94 stars, based on 1 article reviews
    lipofectamine 3000 - by Bioz Stars, 2026-09
    94/100 stars

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    Related Articles

    Plasmid Preparation:

    Article Title: Transition of CELF2 PAS usage promotes recovery of refractory JDM through alternative splicing regulation of CTSB
    Article Snippet: Then validated products were digested with Spe I/Not I sites and cloned into pIS1 vector (Addgene, USA). .. 2 μg plasmid was transfected into SK-N-BE(2) cells using Lipofectamine 3000 (Thermo Fisher Scientific, USA). ..

    Article Title: Antisense oligonucleotides targeting the SARS-CoV-2 nucleocapsid gene decrease viral titers in hamsters
    Article Snippet: .. In short, SARS-CoV-2/Zsgreen was obtained from a pCC1-4K-SARS-CoV-2-ZsGreen plasmid in VeroE6 cells, which were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) as per the manufacturer’s recommendations, and ZsGreen-expressing viral particles were harvested from culture media. .. To engineer a SARS-CoV-2 minigenome reporter, a sequence containing the SARS-CoV-2 5′ UTR followed by the firefly luciferase gene, the SARS-CoV-2 N gene, and the SARS-CoV-2 3′ UTR was synthesized by Genecust, France, and cloned in the pVITRO2-neo-GFP/LacZ (Thermo Fisher Scientific) at BglII-NheI sites, so that the Luc and N genes were on the same transcript but not in frame ( ).

    Article Title: A critical signaling role for diacylglycerol in phagocytosis of M. tuberculosis
    Article Snippet: Plasmids were extracted from bacterial cultures using Omega E.Z.N.Z Plasmid DNA Mini Kit miniprep kit (Omega Bio-Tek, product D6945-02) following manufacturer instructions. .. Replication deficient lentivirus was produced in HEK 293T cells (ATCC, product CRL-3216) by transfection of the complete lentiCRISPRv2 plasmid, psPAX (a gift from Didier Trono, Addgene plasmid #12260), and pMD2.G (a gift from Didier Trono, Addgene plasmid #12259) via Lipofectamine 3000 (ThermoFisher, product L3000015) following manufacturer instructions. ..

    Article Title: Egr-1 promotes the proliferation and migration of vascular smooth muscle cells by transcriptionally activating Egr-2 in arteriovenous fistulas
    Article Snippet: Rat EGR1 promoter (~1.3 Kb; PCR-amplified from rat VSMCs) cloned into pGL3-basic luciferase rep plasmid (Promega Corporation) to form EGR1 luciferase reporters. .. Briefly, VSMCs were (3×10 4 /well in 24-well plates) seeded and incubated for 24 h. Specific plasmids, lentivirus and 1.5 ng of pRL-TK Renilla plasmid were transfected with Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). .. At 24 h after transfection, luciferase and Renilla signals were determined using the Dual luciferase reporter gene assay kit (cat. no. RG027; Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

    Transfection:

    Article Title: Transition of CELF2 PAS usage promotes recovery of refractory JDM through alternative splicing regulation of CTSB
    Article Snippet: Then validated products were digested with Spe I/Not I sites and cloned into pIS1 vector (Addgene, USA). .. 2 μg plasmid was transfected into SK-N-BE(2) cells using Lipofectamine 3000 (Thermo Fisher Scientific, USA). ..

    Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer
    Article Snippet: Fluorescence images were acquired with a fluorescence microscope (Olympus IX73, Olympus Corporation, Tokyo, Japan), and the number of EdU-positive proliferating cells was quantified for statistical analysis. .. RM-1 cells were transfected with Trp53 -specific siRNA (sense: 5′-GAAUGAGGCCUUAGAGUUATT-3′; antisense: 5′-UAACUCUAAGGCCUCAUUCTT-3′) via Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Carlsbad, California, USA) according to the manufacturer’s protocol (see Table S2). .. After 48 h, the cells were harvested, lysed in radioimmunoprecipitation assay (RIPA; Beyotime Biotechnology, Shanghai, China) buffer supplemented with protease inhibitors, and subjected to Western blotting as described below.

    Article Title: Characterization and functional analysis of BRCA1 and BRCA2 variants in a cohort of 100 unselected patients undergoing germline screening.
    Article Snippet: .. Transient transfection was performed on both MCF7 and MDA-MB-231 cells using lipofectamine 3000 (Thermo Fisher Scientific, Wilmington, DE, USA) according to the manufacturer’s instructions. ..

    Article Title: Antisense oligonucleotides targeting the SARS-CoV-2 nucleocapsid gene decrease viral titers in hamsters
    Article Snippet: .. In short, SARS-CoV-2/Zsgreen was obtained from a pCC1-4K-SARS-CoV-2-ZsGreen plasmid in VeroE6 cells, which were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) as per the manufacturer’s recommendations, and ZsGreen-expressing viral particles were harvested from culture media. .. To engineer a SARS-CoV-2 minigenome reporter, a sequence containing the SARS-CoV-2 5′ UTR followed by the firefly luciferase gene, the SARS-CoV-2 N gene, and the SARS-CoV-2 3′ UTR was synthesized by Genecust, France, and cloned in the pVITRO2-neo-GFP/LacZ (Thermo Fisher Scientific) at BglII-NheI sites, so that the Luc and N genes were on the same transcript but not in frame ( ).

    Article Title: A critical signaling role for diacylglycerol in phagocytosis of M. tuberculosis
    Article Snippet: Plasmids were extracted from bacterial cultures using Omega E.Z.N.Z Plasmid DNA Mini Kit miniprep kit (Omega Bio-Tek, product D6945-02) following manufacturer instructions. .. Replication deficient lentivirus was produced in HEK 293T cells (ATCC, product CRL-3216) by transfection of the complete lentiCRISPRv2 plasmid, psPAX (a gift from Didier Trono, Addgene plasmid #12260), and pMD2.G (a gift from Didier Trono, Addgene plasmid #12259) via Lipofectamine 3000 (ThermoFisher, product L3000015) following manufacturer instructions. ..

    Article Title: Egr-1 promotes the proliferation and migration of vascular smooth muscle cells by transcriptionally activating Egr-2 in arteriovenous fistulas
    Article Snippet: Rat EGR1 promoter (~1.3 Kb; PCR-amplified from rat VSMCs) cloned into pGL3-basic luciferase rep plasmid (Promega Corporation) to form EGR1 luciferase reporters. .. Briefly, VSMCs were (3×10 4 /well in 24-well plates) seeded and incubated for 24 h. Specific plasmids, lentivirus and 1.5 ng of pRL-TK Renilla plasmid were transfected with Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). .. At 24 h after transfection, luciferase and Renilla signals were determined using the Dual luciferase reporter gene assay kit (cat. no. RG027; Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

    Article Title: Lapatinib induces ferroptosis in osteosarcoma via the SLC1A5-GPX4 axis
    Article Snippet: .. Cells were seeded in 6-well plates (5 × 10 5 cells/well) and transfected with 50 nM siRNA using Lipofectamine 3000 (Invitrogen, USA) for 8 h. After 48 h of transfection, cells were treated with or without lapatinib (5 μM) for 24 h. Knockdown efficiency was confirmed by Western blot ( J). ..

    Multiple Displacement Amplification:

    Article Title: Characterization and functional analysis of BRCA1 and BRCA2 variants in a cohort of 100 unselected patients undergoing germline screening.
    Article Snippet: .. Transient transfection was performed on both MCF7 and MDA-MB-231 cells using lipofectamine 3000 (Thermo Fisher Scientific, Wilmington, DE, USA) according to the manufacturer’s instructions. ..

    Produced:

    Article Title: A critical signaling role for diacylglycerol in phagocytosis of M. tuberculosis
    Article Snippet: Plasmids were extracted from bacterial cultures using Omega E.Z.N.Z Plasmid DNA Mini Kit miniprep kit (Omega Bio-Tek, product D6945-02) following manufacturer instructions. .. Replication deficient lentivirus was produced in HEK 293T cells (ATCC, product CRL-3216) by transfection of the complete lentiCRISPRv2 plasmid, psPAX (a gift from Didier Trono, Addgene plasmid #12260), and pMD2.G (a gift from Didier Trono, Addgene plasmid #12259) via Lipofectamine 3000 (ThermoFisher, product L3000015) following manufacturer instructions. ..

    Article Title: YTHDF2 regulates the adriamycin (ADM) resistance in NKTCL by modulating the m6A modification of SLC16A9
    Article Snippet: .. Lentiviral particles were produced by co-transfecting the expression vectors with packaging and envelope plasmids into 293T cells using Lipofectamine 3000 (Thermo Fisher Scientific, USA). .. Viral supernatants were collected, filtered, and used to infect NKYS and NK92 cells in the presence of 8 μg/mL polybrene (MilliporeSigma, USA).

    Incubation:

    Article Title: Egr-1 promotes the proliferation and migration of vascular smooth muscle cells by transcriptionally activating Egr-2 in arteriovenous fistulas
    Article Snippet: Rat EGR1 promoter (~1.3 Kb; PCR-amplified from rat VSMCs) cloned into pGL3-basic luciferase rep plasmid (Promega Corporation) to form EGR1 luciferase reporters. .. Briefly, VSMCs were (3×10 4 /well in 24-well plates) seeded and incubated for 24 h. Specific plasmids, lentivirus and 1.5 ng of pRL-TK Renilla plasmid were transfected with Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). .. At 24 h after transfection, luciferase and Renilla signals were determined using the Dual luciferase reporter gene assay kit (cat. no. RG027; Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

    Knockdown:

    Article Title: Lapatinib induces ferroptosis in osteosarcoma via the SLC1A5-GPX4 axis
    Article Snippet: .. Cells were seeded in 6-well plates (5 × 10 5 cells/well) and transfected with 50 nM siRNA using Lipofectamine 3000 (Invitrogen, USA) for 8 h. After 48 h of transfection, cells were treated with or without lapatinib (5 μM) for 24 h. Knockdown efficiency was confirmed by Western blot ( J). ..

    Western Blot:

    Article Title: Lapatinib induces ferroptosis in osteosarcoma via the SLC1A5-GPX4 axis
    Article Snippet: .. Cells were seeded in 6-well plates (5 × 10 5 cells/well) and transfected with 50 nM siRNA using Lipofectamine 3000 (Invitrogen, USA) for 8 h. After 48 h of transfection, cells were treated with or without lapatinib (5 μM) for 24 h. Knockdown efficiency was confirmed by Western blot ( J). ..

    Expressing:

    Article Title: YTHDF2 regulates the adriamycin (ADM) resistance in NKTCL by modulating the m6A modification of SLC16A9
    Article Snippet: .. Lentiviral particles were produced by co-transfecting the expression vectors with packaging and envelope plasmids into 293T cells using Lipofectamine 3000 (Thermo Fisher Scientific, USA). .. Viral supernatants were collected, filtered, and used to infect NKYS and NK92 cells in the presence of 8 μg/mL polybrene (MilliporeSigma, USA).



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    PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by <t>Lipofectamine</t> <t>3000</t> (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .
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    PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by <t>Lipofectamine</t> <t>3000</t> (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .
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    PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by <t>Lipofectamine</t> <t>3000</t> (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .
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    PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by <t>Lipofectamine</t> <t>3000</t> (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .
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    Image Search Results


    PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by Lipofectamine 3000 (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .

    Journal: Nucleic Acids Research

    Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

    doi: 10.1093/nar/gkag328

    Figure Lengend Snippet: PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by Lipofectamine 3000 (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .

    Article Snippet: As we observed that activation levels by polyRNAs in different reporter cell lines are influenced by the transfection reagent, we used either the commercial lipid-based Lipofectamine 3000 or polymer-based Mirus TransIT-X2 transfection reagent, as optimized for each cell line.

    Techniques: Activation Assay, In Vitro, Transfection, Expressing, High Molecular Weight, Standard Deviation

    LNP formulation can be optimized for polyRNAs of varying lengths. ( A ) Structures of polyRNAs used for LNP formulation screening. ( B ) Size distributions of polyRNAs, as analysed by agarose gel electrophoresis. The average length of each polyRNA was determined using ImageJ. T3, T52, T43, and T51 polyRNAs comprise hairpin repeat motifs, while T19 polyRNA comprises nonhairpin, predominantly single-stranded repeats. ( C, D ) Activation of the IRF pathway by polyRNAs delivered by Lipofectamine 3000 (Lipo) and LNPs in RAW-Dual reporter cells, shown as absolute values ( C ) and as percentages of Lipofectamine/polyRNA IRF activation ( D ). ( E, F ) Activation of the NF-κB pathway by polyRNAs delivered by Lipofectamine 3000 (Lipo) and MC3-based LNPs in RAW-Dual reporter cells, shown as absolute values ( E ) and as percentages of Lipofectamine/polyRNA NF-κB activation ( F ). All dosages of polyRNA and the poly(I:C) benchmark were 0.5 µg/ml. MC3 lipid from MedChemExpress was used for LNP synthesis in this figure. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way ANOVA with Šidak’s multiple comparisons test. ns, no significant difference between bracketed groups. */**/***/**** denotes significance between bracketed groups ( P <.05/.01/.001/.0001). Figure was created in BioRender. Yang, Y. (2026) https://BioRender.com/m44nwii .

    Journal: Nucleic Acids Research

    Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

    doi: 10.1093/nar/gkag328

    Figure Lengend Snippet: LNP formulation can be optimized for polyRNAs of varying lengths. ( A ) Structures of polyRNAs used for LNP formulation screening. ( B ) Size distributions of polyRNAs, as analysed by agarose gel electrophoresis. The average length of each polyRNA was determined using ImageJ. T3, T52, T43, and T51 polyRNAs comprise hairpin repeat motifs, while T19 polyRNA comprises nonhairpin, predominantly single-stranded repeats. ( C, D ) Activation of the IRF pathway by polyRNAs delivered by Lipofectamine 3000 (Lipo) and LNPs in RAW-Dual reporter cells, shown as absolute values ( C ) and as percentages of Lipofectamine/polyRNA IRF activation ( D ). ( E, F ) Activation of the NF-κB pathway by polyRNAs delivered by Lipofectamine 3000 (Lipo) and MC3-based LNPs in RAW-Dual reporter cells, shown as absolute values ( E ) and as percentages of Lipofectamine/polyRNA NF-κB activation ( F ). All dosages of polyRNA and the poly(I:C) benchmark were 0.5 µg/ml. MC3 lipid from MedChemExpress was used for LNP synthesis in this figure. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way ANOVA with Šidak’s multiple comparisons test. ns, no significant difference between bracketed groups. */**/***/**** denotes significance between bracketed groups ( P <.05/.01/.001/.0001). Figure was created in BioRender. Yang, Y. (2026) https://BioRender.com/m44nwii .

    Article Snippet: As we observed that activation levels by polyRNAs in different reporter cell lines are influenced by the transfection reagent, we used either the commercial lipid-based Lipofectamine 3000 or polymer-based Mirus TransIT-X2 transfection reagent, as optimized for each cell line.

    Techniques: Formulation, Agarose Gel Electrophoresis, Activation Assay, Standard Deviation

    Assembly of CpG-DNA motifs onto polyRNA scaffolds induces activation of multiple PRRs in vitro . ( A ) Structure of a CpG-DNA comb, comprising a region complementary to a polyRNA repeat sequence, a linker, and a CpG-DNA sequence. Agarose gel electrophoresis of T52 polyRNA and DNA comb-patterned polyRNA indicates successful hybridization. T52 polyRNA comprises hairpin repeat motifs and averages 1000 bases in length. Complementary DNA comb: D52. The D52 DNA comb was hybridized to T52 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:3. ( C, D ) In vitro activation of the IRF ( C ) and NF-κB ( D ) pathways by T19 polyRNA and its CpG-DNA hybrid, transfected by Lipofectamine 3000 (Lipo) in RAW-Dual reporter cells, compared to the PRR agonist benchmarks poly(I:C) and 5′ triphosphate hairpin RNA (3p-hpRNA). A CpG-DNA comb only quantitative control was dosed at the equivalent amount used for hybridizing to polyRNA. T19 polyRNA comprises nonhairpin repeat motifs and averages 7000 bases in length. The D19-CpG comb is a DNA oligo mTLR9 agonist with a complementary region to T19 repeats and was hybridized to T19 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:4. ( E, F ) In vitro activation of PRRs by T52 polyRNA and its CpG-DNA hybrid, transfected with D-Lin-MC3-DMA (MC3)-based LNPs in RAW-Dual ( E ) and HEK-Blue mTLR9 reporter cells ( F ). D52-CpG is a DNA oligo mTLR9 agonist with a complementary region to T52 repeats. D52.mismatch-CpG is a mismatch comb control that does not contain any region complementary to T52 repeats. CpG-DNA combs were hybridized to T52 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:3. All dosages of polyRNAs, polyRNA/CpG-DNA (dosing based on polyRNA mass), and benchmark PRR agonists were 0.5 µg/ml. All CpG DNA comb-only samples were dosed at levels equivalent to the amounts hybridized to their corresponding base polyRNA scaffolds. The data represent the mean ± standard deviation of n = 3 technical replicates ( C–F ). Data were analysed by one-way ANOVA with Šidak’s multiple comparisons test. ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/99l41mi .

    Journal: Nucleic Acids Research

    Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

    doi: 10.1093/nar/gkag328

    Figure Lengend Snippet: Assembly of CpG-DNA motifs onto polyRNA scaffolds induces activation of multiple PRRs in vitro . ( A ) Structure of a CpG-DNA comb, comprising a region complementary to a polyRNA repeat sequence, a linker, and a CpG-DNA sequence. Agarose gel electrophoresis of T52 polyRNA and DNA comb-patterned polyRNA indicates successful hybridization. T52 polyRNA comprises hairpin repeat motifs and averages 1000 bases in length. Complementary DNA comb: D52. The D52 DNA comb was hybridized to T52 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:3. ( C, D ) In vitro activation of the IRF ( C ) and NF-κB ( D ) pathways by T19 polyRNA and its CpG-DNA hybrid, transfected by Lipofectamine 3000 (Lipo) in RAW-Dual reporter cells, compared to the PRR agonist benchmarks poly(I:C) and 5′ triphosphate hairpin RNA (3p-hpRNA). A CpG-DNA comb only quantitative control was dosed at the equivalent amount used for hybridizing to polyRNA. T19 polyRNA comprises nonhairpin repeat motifs and averages 7000 bases in length. The D19-CpG comb is a DNA oligo mTLR9 agonist with a complementary region to T19 repeats and was hybridized to T19 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:4. ( E, F ) In vitro activation of PRRs by T52 polyRNA and its CpG-DNA hybrid, transfected with D-Lin-MC3-DMA (MC3)-based LNPs in RAW-Dual ( E ) and HEK-Blue mTLR9 reporter cells ( F ). D52-CpG is a DNA oligo mTLR9 agonist with a complementary region to T52 repeats. D52.mismatch-CpG is a mismatch comb control that does not contain any region complementary to T52 repeats. CpG-DNA combs were hybridized to T52 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:3. All dosages of polyRNAs, polyRNA/CpG-DNA (dosing based on polyRNA mass), and benchmark PRR agonists were 0.5 µg/ml. All CpG DNA comb-only samples were dosed at levels equivalent to the amounts hybridized to their corresponding base polyRNA scaffolds. The data represent the mean ± standard deviation of n = 3 technical replicates ( C–F ). Data were analysed by one-way ANOVA with Šidak’s multiple comparisons test. ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/99l41mi .

    Article Snippet: As we observed that activation levels by polyRNAs in different reporter cell lines are influenced by the transfection reagent, we used either the commercial lipid-based Lipofectamine 3000 or polymer-based Mirus TransIT-X2 transfection reagent, as optimized for each cell line.

    Techniques: Activation Assay, In Vitro, Sequencing, Agarose Gel Electrophoresis, Hybridization, Transfection, Control, Standard Deviation