Amaxa nucleofector technology

Manufactured by Lonza

Sourced in Switzerland, Germany, United States, France

The Amaxa Nucleofector Technology is a laboratory equipment product that enables the efficient transfection of various cell types, including difficult-to-transfect primary cells and stem cells. The technology utilizes electrical pulses to facilitate the direct delivery of nucleic acids, such as DNA and RNA, into the cell nucleus.

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Lab products found in correlation

Pcdna3 flag mkk6 glu, by Addgene (4 mentions) On targetplus smartpool sirna, by Horizon Discovery (4 mentions) Sigenome smartpool sirna, by Horizon Discovery (3 mentions) Sigenome smartpool, by Horizon Discovery (3 mentions) On targetplus smartpool, by Horizon Discovery (2 mentions) Scrambled sirna, by Santa Cruz Biotechnology (2 mentions) Mnsod, by Santa Cruz Biotechnology (2 mentions) Pcmv3, by Sino Biological (2 mentions) Basic nucleofector kit, by Lonza (2 mentions) Pcmv3 ocln plasmid, by Sino Biological (2 mentions) 27mer oas1 sirnas, by OriGene (2 mentions) 27mer oas2 sirnas, by OriGene (2 mentions) 27mer oas3 sirnas, by OriGene (2 mentions) 27mer oasl sirnas, by OriGene (2 mentions) 27mer rnasel sirnas, by OriGene (2 mentions) 27mer occludin sirnas, by OriGene (2 mentions) 27mer tjp1 sirnas, by OriGene (2 mentions) Scrambled sirna, by Thermo Fisher Scientific (2 mentions) Edta free protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Incucyte s3 live cell analysis system, by Sartorius (2 mentions)

Close spelling variants of this product

Amaxa Nucleofector (332 citations) Nucleofector Technology (56 citations) Amaxa technology (9 citations)

71 protocols using amaxa nucleofector technology

Modulating Signaling Pathways in MDMs

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100nM scrambled or siGENOME SMARTpool siRNAs (a pool of four distinct, commercially designed siRNA) against IRF5 (#M-011706-00), Akt1 (#M-003000-03), Akt2 (#M-003001-02), NOD2 (#M-003464-01), IKKβ (#M–003503–03), IRAK-1 (#M-004760-03), TRAF6 (#M-004712-00), BCAP (#M-016285-01), RIP2 (#M-003602-02), HK1 (#M-006820-01), TPI1 (#M-009776-02), PGAM (#M-008883-01), or HIF1A (#M-004018-05) (GE Dharmacon) or 4µg pMCL-MKK1 (R4F) (constitutively active ERK kinase)(Mansour et al., 1994 (link)), 4µg pSRα-3HA-JNKK2-JNK1-WT (constitutively active JNK)(Zheng et al., 1999 (link)) (generous gifts from Dr. Ben Turk), 4µg pcDNA3-Flag MKK6(glu) (constitutively active p38 kinase) (Addgene plasmid 13518; kindly deposited by Roger Davis (Raingeaud et al., 1996 (link))), 2µg IKK-2 S177D S181E (constitutively active NFκB) (Addgene plasmid 11105; kindly deposited by Anjana Rao (Mercurio et al., 1997 (link))), 2 µg pcDNA3 Myr HA Akt2 (constitutively active Akt2) (Addgene plasmid 9016, kindly deposited by William Sellars), 2µg IRF5 vector (Genecopoeia, Rockville, MD) or empty vector (a control vector without the gene of interest) were transfected for 48h (unless otherwise indicated) into MDMs using Amaxa nucleofector technology (Amaxa, San Diego, CA). Transfection efficiency was >70%.

Hedl M., Yan J, & Abraham C. (2016). IRF5 and IRF5 Disease-Risk Variants Increase Glycolysis and Human M1 polarization By Regulating Proximal Signaling and Akt2 Activation. Cell reports, 16(9), 2442-2455.

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Assessing NF-κB Activity in Endothelial Cells

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Transcriptional activity of NF-κB was tested in serum-treated detector endothelial cells by a reporter gene assay, to determine cellular pro-inflammatory effects induced by factors in the sera.^{17 (link)} Transfections in endothelial cells were performed using the Amaxa Nucleofector technology (Amaxa, Gaithersburg, MD), as we have previously reported.^{17 (link)}

Gardner A.W., Parker D.E., Montgomery P.S., Sosnowska D., Casanegra A.I., Ungvari Z., Csiszar A, & Sonntag W.E. (2015). ENDOTHELIAL CELL INFLAMMATION AND ANTIOXIDANT CAPACITY ARE ASSOCIATED WITH EXERCISE PERFORMANCE AND MICROCIRCULATION IN PATIENTS WITH SYMPTOMATIC PERIPHERAL ARTERY DISEASE. Angiology, 66(9), 867-874.

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Measuring Phospholipase D Activity in Cells

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In vivo PLD activity was determined by PLD-catalyzed transphosphatidylation as previously described20 (link). Briefly, 3T3-L1 cells were metabolically labeled with ³H-oleic acid for 18 h. After pretreatment with 0.3% 1-butanol for 30 min, cells were lysed and lipids were extracted according to the method of Bligh and Dyer57 (link) and analyzed by TLC. Alternatively, a Phospholipase D Assay Kit (Sigma Aldrich) was also used, as indicated in the figure legends, to measure PLD activity according to the manufacturer’s instructions58 (link)59 (link)60 (link)61 (link). To calculate recombinant PLD1 or PLD2 activity specifically, the PLD activity in cells transfected with empty vector was subtracted from the activity in PLD1 or PLD2-transfected cells under the same conditions20 (link)62 (link). Cells were transfected with HA-PLD120 (link) and HA-PLD220 (link) using Amaxa Nucleofector technology (Amaxa, Cologne, Germany) according to manufacturers’ protocol.

Song H.I, & Yoon M.S. (2016). PLD1 regulates adipogenic differentiation through mTOR - IRS-1 phosphorylation at serine 636/639. Scientific Reports, 6, 36968.

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Macrophage Transfection and Pathway Modulation

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Primary human MDMs were transfected with 200nM scrambled or ON-TARGETplus SMARTpool siRNA against Tyro3, Axl, Mer, Gas6, Protein S, SOCS3, c-Fos, c-Jun, musculoaponeurotic fibrosarcoma oncogene homolog K (MAFK), PU.1 (Dharmacon, Lafayette, CO) (4 pooled siRNAs for each gene), or pReceiver-M2-SOCS3 (GenoCopoeia, Rockville, MD), pMCL-MKK1 (R4F) (constitutively active ERK kinase)(22 (link)), pSRα-3HA-JNKK2-JNK1-WT (constitutively active JNK)(23 (link)) (generous gifts from Dr. Ben Turk), pCDNA3-Flag MKK6(glu) (constitutively active p38 kinase)(24 (link)) (Addgene plasmid 13518) or empty vector using Amaxa nucleofector technology (Amaxa, San Diego, CA).

Zheng S., Hedl M, & Abraham C. (2015). TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to pro-inflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages. Journal of immunology (Baltimore, Md. : 1950), 194(4), 1928-1937.

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NF-κB Transcriptional Activity in Inflamed Endothelial Cells

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Transcriptional activity of NF-κB was tested in serum-treated detector endothelial cells by a reporter gene assay, to determine cellular pro-inflammatory effects induced by factors in the sera.^{8 (link)} Transfections in endothelial cells were performed using the Amaxa Nucleofector technology (Amaxa, Gaithersburg, MD), as we have previously reported.^{8 (link)}

Gardner A.W., Montgomery P.S., Zhao Y.D., Silva-Palacios F., Ungvari Z., Csiszar A, & Sonntag W.E. (2017). ASSOCIATION BETWEEN DAILY WALKING AND ANTIOXIDANT CAPACITY IN PATIENTS WITH SYMPTOMATIC PERIPHERAL ARTERY DISEASE. Journal of vascular surgery, 65(6), 1762-1768.

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Silencing Immune Signaling Molecules

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Primary human MDMs were transfected with 100nM or the indicated doses of scrambled or ON-TARGETplus or siGENOME SMARTpool siRNA against STAT3, STAT5a, STAT5b, IL10RA, TGFBR, IL4RA, IL13RA, IL22RA, CRLF2, SOCS1, SOCS2, SOCS3 (Dharmacon, Lafayette, CO) (4 pooled siRNAs for each gene) using Amaxa nucleofector technology (Amaxa, San Diego, CA).

Hedl M., Sun R., Huang C, & Abraham C. (2019). STAT3 and STAT5 signaling thresholds determine distinct regulation for innate-receptor-induced inflammatory cytokines and STAT3/STAT5 disease variants modulate these outcomes. Journal of immunology (Baltimore, Md. : 1950), 203(12), 3325-3338.

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Efficient siRNA Transfection for Gene Silencing

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Small interfering RNA (siRNA) transfection was performed by using Amaxa Nucleofector technology (Amaxa GmbH, Cologne, Germany), using 0.5 nmol of siRNA against JAK3, STAT5a or STAT5b and non-targeting (NT) control #1 (ONtarget PLUS, smart pool, Dharmacon, Chicargo, IL, USA) or siRNA directed against TNFR2 and universal negative control #1 (sigma) on 2×10⁶ cells, described in detail previously [48 (link)].

Lauenborg B., Christensen L., Ralfkiaer U., Kopp K.L., Jønson L., Dabelsteen S., Bonefeld C.M., Geisler C., Gjerdrum L.M., Zhang Q., Wasik M.A., Ralfkiaer E., Ødum N, & Woetmann A. (2015). Malignant T cells express lymphotoxin α and drive endothelial activation in cutaneous T cell lymphoma. Oncotarget, 6(17), 15235-15249.

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Modulating Myeloid Cell Signaling

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300 nM scrambled or ON-TARGETplus SMARTpool small interfering RNA (siRNA) against IL-18RAP (Dharmacon, Lafayette, CO) (4 pooled siRNAs for each gene) or 5μg pMCL-MKK1 (R4F) (constitutively active ERK kinase)(28 (link)), pSRα-3HA-JNKK2-JNK1-WT (constitutively active JNK)(29 (link)) (generous gifts from Dr. Ben Turk), pCDNA3-Flag MKK6(glu) (constitutively active p38 kinase)(30 (link)) (Addgene plasmid 13518) or empty vector were transfected into myeloid cells using Amaxa nucleofector technology (Amaxa, San Diego, CA). Cells were cultured for an additional 48h and then treated as indicated, or in some cases stained with annexin V-FITC (eBiosciences) to ensure cell viability.

Hedl M., Zheng S, & Abraham C. (2014). The IL18RAP Region Disease Polymorphism Decreases IL-18RAP/IL-18R1/IL-1R1 Expression and Signaling Through Innate Receptor-Initiated Pathways. Journal of immunology (Baltimore, Md. : 1950), 192(12), 5924-5932.

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Serum-induced NF-κB Activation in Endothelial Cells

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To assess cellular proinflammatory effects induced by factors present in the sera, transcriptional activity of NF-κB was tested in serum-treated detector endothelial cells by a reporter gene assay as described [31 ]. Transfections in endothelial cells were performed using the Amaxa Nucleofector technology (Amaxa, Gaithersburg, MD), as we have previously reported [31 ].

Gardner A.W., Parker D.E., Montgomery P.S., Sosnowska D., Casanegra A.I., Ungvari Z., Csiszar A, & Sonntag W.E. (2014). Greater Endothelial Apoptosis and Oxidative Stress in Patients with Peripheral Artery Disease. International Journal of Vascular Medicine, 2014, 160534.

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Measuring NF-κB Activation in Endothelial Cells

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Transcriptional activity of NF-κB was tested in serum-treated detector endothelial cells by a reporter gene assay, to determine cellular pro-inflammatory effects induced by factors in the sera [23] (link). Transfections in endothelial cells were performed using the Amaxa Nucleofector technology (Amaxa, Gaithersburg, MD), as we have previously reported [23] (link).

Gardner A.W., Parker D.E., Montgomery P.S., Sosnowska D., Casanegra A.I., Ungvari Z., Csiszar A., Zhang S.X., Wang J.J, & Sonntag W.E. (2015). Influence of diabetes on ambulation and inflammation in men and women with symptomatic peripheral artery disease. Journal of Clinical & Translational Endocrinology, 2(4), 137-143.

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