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Amaxa nucleofection technology

Manufactured by Lonza
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Amaxa nucleofection technology is a transfection method that enables the delivery of genetic material into a wide range of cell types, including difficult-to-transfect cells. The technology utilizes electrical pulses to temporarily permeabilize the cell membrane, allowing for the efficient transfer of DNA, RNA, or other molecules into the cell's nucleus.

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Lab products found in correlation

Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions) Silencer select sirna, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Muse count viability kit, by Merck Group (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Muse cell analyzer, by Merck Group (1 mentions) Cell line solution kit 5, by Lonza (1 mentions) Nucleofector 2 device, by Lonza (1 mentions) Nucleofector apparatus, by Lonza (1 mentions) Sirna targeting, by Thermo Fisher Scientific (1 mentions) Sirna targeting, by Horizon Discovery (1 mentions) Non targeting control sirna, by Horizon Discovery (1 mentions) Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions) On targetplus control pool, by Horizon Discovery (1 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)

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Amaxa nucleofection Nucleofection technology Amaxa technology Nucleofection

14 protocols using amaxa nucleofection technology

1

Transfection of CLL cells with MCL-1 siRNA

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CLL cells were transfected using the AMAXA nucleofection technology (Amaxa, Cologne, Germany) with one of two different MCL-1 siRNAs or control siRNA according to the manufacturer’s instructions and as described.46 (link) In short, CLL cells were left to recover after thawing for at least 3 h at 37 °C in 5% CO2. Cells (5.5 × 106) were resuspended in 100 μl human B-cell nucleofector kit solution (Lonza, Basel, Switzerland) and nucleofected with Silencer Select siRNAs (final concentration 3 μM) directed against MCL-1 or a negative control (Ambion, Paisley, UK; catalog numbers s8585 (two CLL samples) and s8583 (three CLL samples) for MCL-1 and AM4635 as negative control) using program X-001. After transfection, cells were directly resuspended in pre-warmed IMDM+/+ and plated in six-well plates to recover for at least 1h before commencing stimulation.
van Attekum M.H., Terpstra S., Slinger E., von Lindern M., Moerland P.D., Jongejan A., Kater A.P, & Eldering E. (2017). Macrophages confer survival signals via CCR1-dependent translational MCL-1 induction in chronic lymphocytic leukemia. Oncogene, 36(26), 3651-3660.
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2

BTK Mutant Transfection and Survival Assay

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TMD8 cells were maintained in RPMI1640 at 37°C with 10% fetal calf serum (Mediatech Inc, Manassas, VA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Fisher Scientific, Fairlawn, NJ, USA). For cell transfection with BTK WT, BTKC481S and BTKT316A mutant constructs, Amaxa Nucleofection technology was applied according to the manufacturer's protocols (Amaxa, Cologne, Germany; kit V, Program U-13). To enhance the cell survival following transfection, TMD8 cells were co-cultured with bone marrow stromal cell line NKTert cells in a 24-well plate for the first 24hr. Cells were subsequently transferred into a new plate and ibr or vehicle was then added into the culture. Cell viability was determined with Muse™ Count & Viability kit using Muse Cell Analyzer (Millipore, Hayward, CA).
Sharma S., Galanina N., Guo A., Lee J., Kadri S., Van Slambrouck C., Long B., Wang W., Ming M., Furtado L.V., Segal J.P., Stock W., Venkataraman G., Tang W.J., Lu P, & Wang Y.L. (2016). Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL. Oncotarget, 7(42), 68833-68841.
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3

Jag1 Silencing in CLL Cells

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CLL cells were transfected using the Amaxa nucleofection technology (Amaxa, Cologne, Germany) and the ON-TARGETplus SMARTpool small interfering RNA (siRNA) to Jag1 (siJag1) or ON-TARGETplus siCONTROL nontargeting pool (siCtrl) as negative control (Dharmacon, Lafayette, CO, USA). CLL cells (12 × 106) were resuspended in 100 µl Cell Line Solution Kit V (Lonza Group Ltd, Basel, Switzerland) with 0.25 μM of siJag1 or siCtrl, transferred to the provided cuvettes and transfected with the Amaxa Nucleofector II device (program U-013). Cells were immediately transferred into 12-well plates in complete medium and cultured for 72 h in the presence of 25 ng/ml IL-4. Cells were then examined for Jag1 protein expression to verify the efficiency of silencing, and for cell viability/apoptosis.
De Falco F., Del Papa B., Baldoni S., Sabatini R., Falzetti F., Di Ianni M., Martelli M.P., Mezzasoma F., Pelullo M., Marconi P., Sportoletti P., Screpanti I, & Rosati E. (2018). IL-4-dependent Jagged1 expression/processing is associated with survival of chronic lymphocytic leukemia cells but not with Notch activation. Cell Death & Disease, 9(12), 1160.
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4

Nucleofection-based Transfection of Cell Lines

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CEMX174, HuT78 cells, and the monkey B-LCLs were transfected using the Amaxa nucleofection technology (Amaxa, Koln, Germany). Cells were suspended in a solution supplied with the Amaxa Human T cell nucleofector kit (VPA-1002 LONZA), following the Amaxa guidelines for cell line transfection (see Amaxa literature for further details about this kit). In brief, 100 μl of a 10 × 106 cell suspension was mixed with either 1 μg pmaxGFP only or combined with 5 μg pGL3-DNA and transferred to the provided cuvette and nucleofected using an Amaxa Nucleofector apparatus (Amaxa). Cells were transfected using the V-001 pulsing parameter and were immediately transferred into tubes containing 1 ml 37°C prewarmed culture medium, and aliquots were equally divided into individual wells of a 12-well plate with or without 1 μM RA. After transfection, cells were cultured for 24–48 h in the case of cell lines and for 6–24 h for the B-LCL, and the cultures were subjected to luciferase measurements. GFP plasmid was used as internal control in the assay to normalize for transfection efficiency. A fraction of cells from the same wells continued in culture for 72 h and were measured using flow cytometry to measure GFP.
A plasmid containing four repeats of an RA response element with luciferase reporter was used as a positive control as separate cuvette to measure RA-induced gene expression.
Byrareddy S.N., Sidell N., Arthos J., Cicala C., Zhao C., Little D.M., Dunbar P., Yang G.X., Pierzchalski K., Kane M.A., Mayne A.E., Song B., Soares M.A., Villinger F., Fauci A.S, & Ansari A.A. (2015). Species-Specific Differences in the Expression and Regulation of α4β7 Integrin in Various Nonhuman Primates. Journal of immunology (Baltimore, Md. : 1950), 194(12), 5968-5979.
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5

Bcl-XL and NIK Knockdown in CLL Cells

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CLL cells were transfected using the Amaxa nucleofection technology (Amaxa) as previously described10 (link) with 3 µg siRNA targeting Bcl-XL and non-targeting siRNA (Ambion, Thermo Fisher Scientific) or 3 µg siRNA targeting NIK and non-targeting siRNA (Dharmacon). After nucleofection, cells were cultured on 3T40L for 24 h before drugs sensitivity assay was performed or protein lysates were obtained.
Haselager M., Thijssen R., West C., Young L., Van Kampen R., Willmore E., Mackay S., Kater A, & Eldering E. (2021). Regulation of Bcl-XL by non-canonical NF-κB in the context of CD40-induced drug resistance in CLL. Cell Death and Differentiation, 28(5), 1658-1668.
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6

Transfection of Primary HSC

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Transfection of primary HSC was performed by Amaxa nucleo-fection technology (Amaxa, Koln, Germany) as previously described [20] , using 100 nM small interfering RNA (siRNA) targeting JNK orTGF-B-B, or control non-targeting siRNA (all provided by Dharma-con Inc., Lafayette, CO). The efficiency of silencing was evaluatedby immunoblotting (JNK) or RT-PCR (TGF-B).
Delogu W., Caligiuri A., Provenzano A., Rosso C., Bugianesi E., Coratti A., Macias-Barragan J., Galastri S., Di Maira G, & Marra F. (2019). Myostatin regulates the fibrogenic phenotype of hepatic stellate cells via c-jun N-terminal kinase activation. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver, 51(10).
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7

Transfection of Human Hematopoietic Stem Cells

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All siRNAs used were purchased from Dharmacon (Lafayette, USA). Transfection of human HSC was performed using the Amaxa nucleofection technology (Amaxa, Koln, Germany) as previously described, with 100 nM smart Pool siRNA specific for human MERTK (sequence accession no. # NM_006343) or non-targeting siRNA [28] .
Petta S., Valenti L., Marra F., Grimaudo S., Tripodo C., Bugianesi E., Cammà C., Cappon A., Di Marco V., Di Maira G., Dongiovanni P., Rametta R., Gulino A., Mozzi E., Orlando E., Maggioni M., Pipitone R.M., Fargion S, & Craxì A. (2016). MERTK rs4374383 polymorphism affects the severity of fibrosis in non-alcoholic fatty liver disease. Journal of hepatology, 64(3).
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8

Knockdown Analysis of Splicing Factors

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CHO-K1 cells (2 × 105) were seeded in 24-well plates and cultured for 24 h. siRNAs targeting SRp20 (NM_003017), SRSF1 (NM_006924.4), and hnRNP A1 (NM_031157.2) (Fig. 6A, B, and C) were transfected into CHO-K1 cells using Amaxa Nucleofection technology (Lonza) following the manufacturer’s recommendations. Cells were then recovered and seeded in pre-warmed 24-well plates. After 24 h incubation, cells were used for ATP7B exon12 exclusion analysis (Fig. 4).
Lin Y.J., Ho T.J., Lin T.H., Hsu W.Y., Huang S.M., Liao C.C., Lai C.H., Liu X., Tsang H., Lai C.C, & Tsai F.J. (2015). P-coumaric acid regulates exon 12 splicing of the ATP7B gene by modulating hnRNP A1 protein expressions. BioMedicine, 5(2), 10.
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9

Transient Transfection Assay for ATP7B Exon12

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For cDNA plasmid transfection assays, CHO-K1 cells (2 × 105) were seeded in 24-well plates and cultured for 24 h. Cells were transiently transfected with the wild-type expression plasmids, pSRp20, pSRSF1, and phnRNP A1, or with the pcDNA3.0 expression plasmid as a mock control using Amaxa Nucleofection technology (Lonza) following the manufacturer’s recommendations. Cells were then recovered and seeded in pre-warmed 24- well plates. After 24 h incubation, cells were used for ATP7B exon12 exclusion analysis (Fig. 5).
Lin Y.J., Ho T.J., Lin T.H., Hsu W.Y., Huang S.M., Liao C.C., Lai C.H., Liu X., Tsang H., Lai C.C, & Tsai F.J. (2015). P-coumaric acid regulates exon 12 splicing of the ATP7B gene by modulating hnRNP A1 protein expressions. BioMedicine, 5(2), 10.
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10

Measuring STAT3 Transcriptional Activity

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The activity of STAT3 was measured by dual luciferase reporter (DLR) assays as described before [19 (link)]. Site-directed mutagenesis to generate STAT3 mutants harboring amino acid exchanges Y705F, S727A, or Y705F/S727A was performed on wild-type STAT3 cDNA. The integrity of all constructs was confirmed by sequencing. For expression, all STAT3 constructs were ligated in frame into the expression vector pmaxKS, encoding a C-terminal HA peptide tag. Transfection into LS411N cells was done using the Amaxa nucleofection technology (Lonza).
Koerdel K., Spitzner M., Meyer T., Engels N., Krause F., Gaedcke J., Conradi L.C., Haubrock M., Beißbarth T., Leha A., Johnsen S.A., Ghadimi B.M., Rose-John S., Grade M, & Wienands J. (2021). NOTCH Activation via gp130/STAT3 Signaling Confers Resistance to Chemoradiotherapy. Cancers, 13(3), 455.
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