Amaxa nucleofector 1

Manufactured by Lonza

Sourced in Germany, United States

The Amaxa Nucleofector I is a compact, robust, and easy-to-use electroporation device designed for efficient transfection of a wide range of cell types, including difficult-to-transfect primary cells and cell lines. It utilizes optimized electrical parameters and proprietary Nucleofector solutions to facilitate the introduction of nucleic acids, proteins, and other molecules into the cell nucleus.

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Lab products found in correlation

Amaxa basic nucleofector kit, by Lonza (2 mentions) 24 well plate, by BD (2 mentions) Human cd34 cells kit, by Lonza (2 mentions) Genesolution sirna, by Qiagen (2 mentions) Vpg 1004, by Lonza (2 mentions) Piggybac transposase, by System Biosciences (2 mentions) Nucleofector solution 5, by Lonza (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Siport neofx transfection agent, by Thermo Fisher Scientific (1 mentions) Silencer select sirna, by Thermo Fisher Scientific (1 mentions) Scrambled sirna, by Merck Group (1 mentions) 40 μm cell strainer, by BD (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Fugene 6, by Promega (1 mentions) Gene pulser, by Bio-Rad (1 mentions) Angiotensin 2, by Merck Group (1 mentions) Fetal calf serum (fcs), by Merck Group (1 mentions) Rpmi 1640, by GE Healthcare (1 mentions) Gene pulser xcell device, by Bio-Rad (1 mentions) Human t cell nucleofector kit, by Lonza (1 mentions)

Spelling variants of this product

Amaxa Nucleofector (332 citations) Nucleofector I (20 citations) Amaxa nucleofector I device (6 citations) Amaxa Human Stem Cell Nucleofector Kit 1 (4 citations)

20 protocols using amaxa nucleofector 1

Silencing HSP70 proteins in KSHV cells

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HEK-293T rKSHV.219 cells seeded on 12-well plates were reverse transfected with either 100 nM of the specific Silencer Select siRNA (Life Technologies) or 100 nM AllStars negative control siRNA (Qiagen) using 7 μl of siPORT NeoFX transfection agent (Life Technologies) per transfection. The siRNA ID for Hsc70 and iHsp70 were s6985 and s6968 respectively. s6968 siRNA targets the two major iHsp70 proteins (HSP70-1 and HSP70-2). Two days post-transfection, cells were transfected again in the same manner. Four days after the first transfection, cells were reactivated and incubated for the desired time. Proteins and total RNA were isolated with TRIzol (Life Technologies) and subsequent Western blot and qRT-PCR were performed.
8 × 10⁶ TREx BCBL1-RTA cells were transfected once with 100 μl of Nucleofector solution V (Lonza) to which 2 μM siRNA (scramble or Hsc70) was added. In addition, to monitor transfection efficiency, 1 μg of the control plasmid pmaxGFP was also co-transfected. Cells were transfected using program T-01 of an Amaxa nucleofector I (Lonza). After nucleofection cells were maintained in six-well plates. Medium was freshly replaced every day.

Baquero-Pérez B, & Whitehouse A. (2015). Hsp70 Isoforms Are Essential for the Formation of Kaposi’s Sarcoma-Associated Herpesvirus Replication and Transcription Compartments. PLoS Pathogens, 11(11), e1005274.

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Knockdown of ERK1 and ERK2 in Monocytes

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In knockdown experiments, small interfering RNA (siRNA) against ERK1 and scrambled siRNA were purchased from Sigma-Aldrich while Signalsilencer ® p42 MAPK (ERK2) siRNA II was purchased from Cell Signaling Company. siRNAs were delivered in monocytes by Amaxa nucleofector I (Lonza). 5′ –GACCGGAUGUUAACCUUUA-3′ and 5′-AAGCUGACCCUGAAGUUCA-3′ sequences were used to knock-down ERK1 and as scrambled control (32 (link),33 (link)). For nucleofection, 5×10⁶ monocytes were re-suspended in 100 μl of nucleofection solution containing 150 pmol siRNA for ERK1 and scrambled control, while siRNA against ERK2 was used according the manufacturer protocol. Nucleofection was performed with the Y-01 program. Immediately after nucleofection, monocytes were resuspended in RPMI medium supplemented with 10% FBS and left to recover overnight in polypropylene culture tubes to avoid adherence. The next morning, monocytes were counted with trypan blue showing that 90% of cells were viable. Then cells were treated with 1 μg/ml LPS for 30 minutes followed by 5 mM ATP for another 30 minutes. Released IL-18 in cell culture medium was measured using ELISA while cells were lysed and analyzed for proteins.

Ghonime M.G., Shamaa O.R., Das S., Eldomany R.A., Fernandes-Alnemri T., Alnemri E.S., Gavrilin M.A, & Wewers M.D. (2014). Inflammasome priming by LPS is dependent upon ERK signaling and proteasome function. Journal of immunology (Baltimore, Md. : 1950), 192(8), 3881-3888.

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Transfection and Autophagy Assays in Cells

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Human siRNA (Integrated DNA Technologies) transfections were done as described previously (Miettinen and Björklund, 2014 (link)), with the exception of HUVECs, which were transfected using Amaxa Nucleofector I (Lonza) according to the supplier’s recommendations and co-transfected GFP as a marker for transfected cells. Non-targeted NC1 siRNA (Integrated DNA Technologies) was used as a control. Drosophila dsRNAs were prepared and used as before (Miettinen et al., 2014 (link)). All siRNA treatments were 40 nM in total (20 + 20 nM when combining two independent siRNAs), with the exceptions of HUVEC cell experiments, where a total of 25 nM siRNAs was used, and MEF experiments, where a total of 80 nM siRNAs was used. Mevalonate was supplemented to the cells as mevalolactone (Sigma-Aldrich). See Table S1 for suppliers and solvents of all small molecule inhibitors and metabolites. See Table S2 for details on siRNAs and dsRNAs.
For expression of wild-type human RAB11A, CA Q70A RAB11A, or GFP-RAB11A, synthetic DNA constructs were cloned into cytomegalovirus promoter-driven Gateway expression vector and transfected as in Miettinen and Björklund (2014) (link). The RFP-GFP-LC3B tandem sensor (Life Technologies) was transfected using BacMam 2.0 expression technology according to the supplier’s recommendations 24 hr before fixation and analysis.

Miettinen T.P, & Björklund M. (2015). Mevalonate Pathway Regulates Cell Size Homeostasis and Proteostasis through Autophagy. Cell Reports, 13(11), 2610-2620.

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Measuring NHEJ and HR Repair Efficiency

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hNCC were stably transfected with either NHEJ reporter cassette or HR reporter cassette by nucleofection with Amaxa Nucleofector-I (Lonza program #W-01) using Amaxa Basic Nucleofector Kit for Primary Mammalian Epithelial Cells. These cassettes contain the GFP gene with recognition sequences for I-SceI endonucleases for induction of DSBs. Stably transfected cells were selected for 10 days with 1 mg/mL of G418, co-transfected (by nucleofection) with 5 μg plasmid encoding I-SceI endonuclease to induce DSBs, and GH pcDNA 3.1 or pcDNA 3.1 vector and 0.5 μg plasmid encoding DsRed (pDsRed2-N1) to control for transfection efficiency. Intact reporters were negative for GFP. Upon induction of a DSB by I-SceI digestion, the functional GFP gene was reconstituted. At 48–72h after nucleofection, the number of GFP-positive cells (corresponding to efficiency of DNA DSB repair) and DsRed-positive cells (indicating efficiency of transfection) was determined by FACS, and the ratio between GFP-positive and DsRed-positive cells used as a measure of DSB repair efficiency. NHEJ repair efficiency was 0.77–0.83 in hNCC and HR efficiency was approximately 0.05 in control cells, consistent with other reports. For each nucleofection, a minimum of 50,000 cells was analyzed by FACS and final data analysis performed using FlowJo software. Experiments were repeated at least 3 times.

Chesnokova V., Zonis S., Apostolou A., Estrada H.Q., Knott S., Wawrowsky K., Michelsen K., Ben-Shlomo A., Barrett R., Gorbunova V., Karalis K, & Melmed S. (2021). Local non-pituitary growth hormone is induced with aging and facilitates epithelial damage. Cell reports, 37(11), 110068.

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Electroporation of Hematopoietic Progenitors

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Hematopoietic progenitor cells were electroporated with CRISPR-Cas9 plasmids, as previously described, using Amaxa Nucleofector I (Lonza) and following the manufacturer’s protocols. Cells were electroporated in cuvettes (electroporation volume of 100 μL) using a human CD34⁺ cells kit (Lonza) and the U08 electroporation program. The total amount of plasmid used was 11.26 μg; hence, when two plasmids were electroporated at the same time, 5.63 μg of each was added to the mix. After electroporation, cells were cultured on a 24-well plate (Falcon) at 2 mL of final volume. In the case of the experiments with selection based on ZsGreen reporter expression, sorting was performed 24 h after electroporation. Aggregates were eliminated before sorting using a 40-μm cell strainer (Falcon).

López-Manzaneda S., Ojeda-Pérez I., Zabaleta N., García-Torralba A., Alberquilla O., Torres R., Sánchez-Domínguez R., Torella L., Olivier E., Mountford J., Ramírez J.C., Bueren J.A., González-Aseguinolaza G, & Segovia J.C. (2020). In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9. Molecular Therapy. Methods & Clinical Development, 19, 426-437.

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siRNA Electroporation of Cell Lines

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For siRNA electroporation, 1 × 10⁶ cells were mixed with 5 μl of 20 μM siRNAs (GeneSolution siRNA, Qiagen; Table 3) and 100 μl of Ingenio electroporation solution (MIR50114; Mirus) and nucleofected with an Amaxa Nucleofector I (Lonza) using program C-13 for PHGGs or T-20 for astrocytes. The efficiency of downregulation was validated using Western blot.

Kim D., Olson J.M, & Cooper J.A. (2024). N-cadherin dynamically regulates pediatric glioma cell migration in complex environments. The Journal of Cell Biology, 223(6), e202401057.

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Transfection of HT1080 and HeLa Cells

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HT1080 fibrosarcoma cells and derivatives and HeLa cells were grown as previously described (72 (link)). Supplementary Material, Table S1 summarizes key properties of various HT1080 derivatives used. For electroporations, a Gene Pulser (BioRad) was used as previously described (72 (link)). For lipofections, lipofectamine 2000 (LF; Life Technologies) or Fugene 6 (Promega) was used according to the manufacturers’ instructions, in multiwall well formats with, per cm², 0.5–1 million cells, a total of 0.4 µg (LF) or 0.6 µg (Fugene) DNA and 1 µl (LF) or 1.8 µl (Fugene) cationic lipid reagent. Under these conditions, 70–90% of HT1080 cells lipofected with a green fluorescent protein (GFP) expression plasmid became GFP⁺ 24–48 h post-lipofection. For nucleofection, an Amaxa Nucleofector I (Lonza) was used according to the manufacturer's recommendations (program L005, transfection solution T).

Gravells P., Ahrabi S., Vangala R.K., Tomita K., Brash J.T., Brustle L.A., Chung C., Hong J.M., Kaloudi A., Humphrey T.C, & Porter A.C. (2015). Use of the HPRT gene to study nuclease-induced DNA double-strand break repair. Human Molecular Genetics, 24(24), 7097-7110.

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Evaluating CACNA1H Mutant Effects

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The plasmids expressing the wild-type CACNA1H as well as CACNA1H p.V1937M mutation were constructed into the pIRES-EGFP-puro vector (Addgene plasmid #45567) using PCR-assisted, site-directed mutagenesis. We used PCR-based direct sequencing to confirm the mutation was successfully cloned into the vector. To evaluate the effect of mutant CACNA1H on the expression of CYP11B2 and aldosterone secretion, the HAC15 cells were transfected with pIRES-EGFP-wild-type CACNA1H or pIRES-EGFP CACNA1H p.V1937M using an Amaxa Nucleofector I (Lonza; 3 million cells, 3 µg of plasmid DNA; program X-005) according to the manufacturer’s instructions. The pIRES-EGFP empty vector was used as a control. After transfection, HAC15 cells were seeded at a density of 1 × 10⁶ cells/well into a 6-well plate. The cells were exposed to 10 nM angiotensin II (Sigma-Aldrich, St. Louis, MO, USA) 48 hours after transfection. The culture supernatant was collected 72 hours after transfection for measuring the concentrations of aldosterone, and cells were harvested for Western blot analysis.

Tseng C.S., Peng K.Y., Wang S.M., Tsai Y.C., Huang K.H., Lin W.C., Hu Y.H., Wu V.C, & Chueh J.S. (2022). A Novel Somatic Mutation of CACNA1H p.V1937M in Unilateral Primary Hyperaldosteronism. Frontiers in Endocrinology, 13, 816476.

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Optimized Transfection of Primary T Cells and Jurkat Cells

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Primary human CD4⁺ T cells were transfected directly after isolation using the Amaxa Nucleofector I device and the human T cell Nucleofector Kit (#VPA-1002, Lonza). Transfection was performed according to the instructions of the manufacturer (program V24, 5μg vector DNA or 300nM small interfering RNA (siRNA), respectively). Jurkat E6.1 T lymphocytes (1x10⁷ per sample) were transfected using the Gene Pulser X cell device equipped with a CE module (Bio-Rad) and 4mm cuvettes (Bridge). Transfection with 10–30μg plasmid DNA was carried out in 500μl RPMI-1640 (GE Healthcare) with 50% FCS (Sigma) using an exponential protocol (240V, 1500μF). Cells were used for functional assays 24h (transfection with DNA) or 48h (transfection with siRNA) later and cultured without antibiotics post-transfection. RNA interference was performed using oligonucleotides with the following sense sequences:
5’-GGUGCCUGUAGGUGAUCAA(dTdT)-3’ (human Dynamin2, oligo #1),5’-GCACUCUGUAUUCUAUUAA(dTdT)-3’ (human Dynamin2, oligo #2),5’-AAACAUGCAGAAAAUGCUG(dTdT)-3’ (Renilla luciferase, control siRNA).All siRNAs were purchased from Quiagen or Dharmacon RNA Technologies, respectively.

Eppler F.J., Quast T, & Kolanus W. (2017). Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion. PLoS ONE, 12(3), e0172443.

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siRNA Nucleofection for Cell Lines

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For siRNA electroporation, 1X10⁶ cells were mixed with 5 μl of 20 μM siRNAs (GeneSolution siRNA, Qiagen) and 100 μl of Ingenio electroporation solution (Mirus, MIR50114) and nucleofected with an Amaxa Nucleofector I (Lonza) using program C-13 for PHGGs or T-20 for astrocytes. The efficiency of downregulation was validated using Western blot.

Kim D., Olson J.M, & Cooper J.A. (2024). N-cadherin dynamically regulates pediatric glioma cell migration in complex environments. bioRxiv.

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