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Nucleofector system

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Sourced in Switzerland, Germany, United States

The Nucleofector system is a lab equipment designed for the transfection of nucleic acids, such as plasmid DNA, siRNA, or mRNA, into various cell types. The system utilizes an electrical pulse to facilitate the efficient delivery of these molecules into the cells. The Nucleofector system is a versatile tool that can be used for a wide range of cell types, including primary cells, stem cells, and difficult-to-transfect cell lines.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Poly l lysine (pll), by Merck Group (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Control sirna, by Thermo Fisher Scientific (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Horse serum, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Fujifilm (2 mentions) Fetal bovine serum (fbs), by Nichirei Biosciences (2 mentions) Pspcas9 bb 2a gfp px458, by Addgene (2 mentions) Cell line nucleofector kit 5, by Lonza (2 mentions) P3 primary cell nucleofector solution and supplement, by Lonza (2 mentions) Effectene kit, by Qiagen (2 mentions) Bj 5ta, by American Type Culture Collection (2 mentions) 2mml glutamine, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Aosmc, by Lonza (2 mentions) Nhost, by Lonza (2 mentions) Smgm 2 bulletkit, by Lonza (2 mentions)

Close spelling variants of this product

4D-Nucleofector system (284 citations) Amaxa Nucleofector system (109 citations) Amaxa 4D-Nucleofector system (45 citations) Amaxa Nucleofector II system (22 citations) Nucleofector II system (16 citations) Nucleofector 96-well Shuttle system (10 citations) Nucleofector 2b system (10 citations) Amaxa Nucleofector 96-well Shuttle System (6 citations) 4D-Nucleofector X Unit system (5 citations) Nucleofector transfection system (4 citations)

80 protocols using nucleofector system

1

Knockdown of BRCA1 and BRCA2 in MCF-7 Cells

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BRCA1 and BRCA2 small hairpin RNAs (shRNAs) were generated using a lentiviral pLKO.1-puromycin vector (Sigma Aldrich). The sequences used for the small hairpin RNA complementary to BRCA1 and BRCA2 were as follows:
For establishment of clonally-derived cell lines, MCF-7 cells were electroporated using nucleofector system (Amaxa) according to manufacturer’s directions. The transfected cells were seeded in 10-cm dishes containing complete DMEM media. After 72h, the plates were washed with PBS and media containing 3μg/ml puromycin was added. The individual puromycin-resistant cells were allowed to grow for another 4 weeks to form colonies. The colonies were picked by placing cloning cylinders around clearly separate colonies. Immunoblotting was used to select the BRCA1 and BRCA2 depleted clones. Clones were maintained in a media containing 3μg/ml puromycin. For transient transfection, we used 4μg of XRCC1, FEN1 siGenome SMART pool siRNA and Non-Targeting siGenome siRNA as a control (Thermo Scientific). Cells were electroporated using the nucleofector system (Amaxa), and were harvested 48h post electroporation.
Fridlich R., Annamalai D., Roy R., Bernheim G, & Powell S.N. (2015). BRCA1 and BRCA2 protect against oxidative DNA damage converted into double-strand breaks during DNA replication. DNA repair, 30, 11-20.
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2

Modulating Actin Dynamics via siRNA and Plasmids

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Cells were transfected with small interfering RNA (siRNA) or plasmids three days before the experiments using a Nucleofector ™ System (Amaxa Biosystems) set to the U25 program. To downregulate Nox4 or SSH1L phosphatase, 3 μg custom designed siRNA against each human sequence (5′-GCAUCUGUUCUUAACCUCA-3′ for Nox4 and 5′-ACUGAGGUACAGCUGGAUGUU-3′ for SH1L) was used to electroporate one million cells. All Stars negative control siRNA (Qiagen 1027281) was used as a control siRNA. To increase cofilin activity, cells were co-transfected with siRNA (negative control or against Nox4) and either 1.1 μg of a plasmid containing a constitutively active form of cofilin (GFP_S3A_cofilin, kind gift of Dr. Condeelis, Albert Einstein College of Medicine, NY) or 1.0 μg of GFP-empty vector as a control. Similarly, in order to intensify LIM kinase activity, cells were transfected with 2 μg of an EGFP-plasmid encoding a constitutively active LIM kinase mutant [6 (link)] or EGFP empty vector (Clontech) as a control.
Montenegro M.F., Valdivia A., Smolensky A., Verma K., Taylor W.R, & Martín A.S. (2015). Nox4-dependent activation of cofilin mediates VSMC reorientation in response to cyclic stretching. Free radical biology & medicine, 85, 288-294.
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3

Generation of Trabecular Meshwork Cell Cultures

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Human tissue for generation of trabecular meshwork cell cultures was obtained from the Eye Bank for Sight Restoration, Inc. (New York, NY, USA) according to the tenets of the Declaration of Helsinki. The study was approved by the Duke University Health System Institutional Review Board. HTM, HDF, and HeLa cells were cultured and maintained following methods previously described.9 (link) Transfections of miRNAs (183 mimic [183M] and negative miRNA control mimic [ConM]) or plasmids (KIAA0101 vector [pKIAA0101] or pcDNA3.1 vector [pCon]; Invitrogen, Carlsbad, CA, USA) to HTM or HDF cells were performed using the Nucleofector system (Amaxa, Inc., Gaithersburg, MD, USA) as previously described.9 (link),10 (link) HeLa cells were transfected using Effectene transfection reagent (Qiagen, Valencia, CA, USA) following the manufacturer instruction.
Li G., Luna C, & Gonzalez P. (2016). miR-183 Inhibits UV-Induced DNA Damage Repair in Human Trabecular Meshwork Cells by Targeting of KIAA0101. Investigative Ophthalmology & Visual Science, 57(4), 2178-2186.
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4

Murine Fibroblast and Myoblast Cell Culture

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NIH3T3 murine fibroblast and C2C12 murine myoblast were maintained at 37°C in 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% calf serum and 10% fetal bovine serum, respectively. To generate myotubes, C2C12 cells were cultured in DMEM supplemented with 2% horse serum (differentiation medium) for five days. Transient transfection of cells with mammalian expression vectors or siRNA was performed using the Nucleofector System (Amaxa GmbH, Cologne, Germany) according to the manufacturer's guidelines (Table 1).
Choi K.Y., Lee M.S., Cho Y.J., Jeong M.H., Han S.J, & Hong S.H. (2014). p104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell. The Scientific World Journal, 2014, 592450.
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5

Efficient Transfection of Porcine Germ Cells

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Example 18

Results are summarized in FIG. 26. Porcine germ cells were isolated from 10 wk old boars, and enriched by differential. Plasmids encoding eGFP and DMD-specific TALENs were transfected into germ cells using the AMAXA NUCLEOFECTOR system Amaxa solutions “V”- and “L” and “B” using programs X-001 and X-005. (SEQ ID NOS: 504, 505, 506) Each transfection reaction was performed with 106 of enriched germ cells, and indicated micrograms of TALEN encoding plasmid DNA. The same methods were used to deliver mRNAs encoding DMD7.1 TALENs. After nucleofection, the cells were cultured for 5 days in 5% CO2 atmosphere at 37° C. or 30° C. Transfection efficiency was evaluated by immunofluorescence analysis for co-localization of expression of GFP and UCH-L1. Cell viability was evaluated by trypan blue exclusion.

US10959415B2. Non-meiotic allele introgression (2021-03-30). Recombinetics, Inc. [US]. Inventors: Scott C. Fahrenkrug [US], Daniel F. Carlson [US].
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6

Efficient Transfection of Porcine Germ Cells

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Example 18

Results are summarized in FIG. 26. Porcine germ cells were isolated from 10 wk old boars, and enriched by differential. Plasmids encoding eGFP and DMD-specific TALENs were transfected into germ cells using the AMAXA NUCLEOFECTOR system Amaxa solutions “V”- and “L” and “B” using programs X-001 and X-005. (SEQ ID NOS: 504, 505, 506) Each transfection reaction was performed with 106 of enriched germ cells, and indicated micrograms of TALEN encoding plasmid DNA. The same methods were used to deliver mRNAs encoding DMD7.1 TALENs. After nucleofection, the cells were cultured for 5 days in 5% CO2 atmosphere at 37° C. or 30° C. Transfection efficiency was evaluated by immunofluorescence analysis for co-localization of expression of GFP and UCH-L1. Cell viability was evaluated by trypan blue exclusion.

US10893667B2. Non-meiotic allele introgression (2021-01-19). Recombinetics, Inc. [US]. Inventors: Scott C. Fahrenkrug [US], Daniel F. Carlson [US].
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7

RNA Isolation and Quantification for miRNA Studies

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Total RNA was isolated using an RNeasy kit (Qiagen) following the manufacturer's protocol. RNA yields were measured using RiboGreen fluorescent dye (Molecular Probes, Eugene, OR, USA). Briefly, HTM cells were transfected with miR-183 mimic (183M) or scramble control mimic (ConM), using the Nucleofector system (Amaxa, Inc.). Three days after transfection, HTM cells were harvested by adding lysis buffer from the kit and homogenization using a QIAshredder spin column (Qiagen). DNA was removed using DNase I (Qiagen). RNA was selectively isolated from the column flow through use of an RNeasy mini-spin column (Qiagen). Twenty microliters of RNase-free water was added to the spin column to recover RNA for each sample. The RNA quantity was analyzed using the RNA Nanodrop with the Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA).
Li G., Luna C, & Gonzalez P. (2016). miR-183 Inhibits UV-Induced DNA Damage Repair in Human Trabecular Meshwork Cells by Targeting of KIAA0101. Investigative Ophthalmology & Visual Science, 57(4), 2178-2186.
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8

Knockdown of REGγ in PC12 ARQ112 cells

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For REGγ knockdown studies: PC12 ARQ112 cell pellets (4 × 106 cells) were resuspended in 100 μl of Nucleofector solution V (Amaxa®) (Lonza Cologne GmbH) with 50 pmol REGγsiRNA (ON-TARGETplus SMART pool rat PSME3), 50 pmol Non-Targeting siRNA (ON-TARGETplus Non-Targeting siRNA #1) (Dharmacon) or 2 μg GFP plasmid (Amaxa). Cells were electrophoresed using the Amaxa Nucleofector System (program U-029) and then replated in 6-well and 24-well dishes. At 72 h post nucleofection, cells were treated with Dox and DHT for 48 h. Cells were harvested at 120 h total and either lysed in Triton DOC lysis buffer and run on SDS-PAGE gels or immunostained. Percentage of siRNA protein knockdown was determined by densitometry. Cells were counted and percent of cells with nuclear inclusions was calculated. Statistical analysis was carried out using the Student’s t-test.
Yersak J.M., Montie H.L., Chevalier-Larsen E.S., Liu Y., Huang L., Rechsteiner M, & Merry D.E. (2017). The 11S Proteasomal Activator REGγ Impacts Polyglutamine-Expanded Androgen Receptor Aggregation and Motor Neuron Viability through Distinct Mechanisms. Frontiers in Molecular Neuroscience, 10, 159.
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9

CRISPR/Cas9-mediated gene knockdown in macrophages and B cells

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Macrophages were transfected by electroporation using the Nucleofector system (Amaxa Biosystems). A total of 5 × 105 cells were suspended in 100 μL of Nucleofector V solution mix (Lonza, VCA‐1003) with 2 μg of GZMA (sc‐437323), RASGRP1 (sc‐437322) CRISPR/Cas9 KO plasmids, or a negative control plasmid harbouring non‐specific guide RNA (SCBT, sc‐418922) was used in nucleofection using the cell line‐specific program T‐O17. CRISPR/Cas9 KO plasmids consist of a pool of three plasmids, each encoding the Cas9 nuclease and a different target‐specific 20 nt guide RNA (gRNA) designed for maximum efficiency. The human B lymphoma cells were transfected with 2 μg of GZMA (SCBT, sc‐403958‐ACT) or RASGRP1 (SCBT, sc‐402120‐ACT) synergistic activation mediator (SAM) transcription activation system consisting of three plasmids encoding the deactivated Cas9 (dCas9) nuclease, the MS2‐p65‐HSF1 fusion protein and a target‐specific 20 nt guide RNA. A non‐specific control plasmid with a non‐specific guide RNA was used as a control (SCBT, sc‐437275). The transfection was done using the T‐O17 program. After transfection, cells were suspended in fresh complete medium and incubated at 37°C with 5% CO2.
Rchiad Z., Haidar M., Ansari H.R., Tajeri S., Mfarrej S., Ben Rached F., Kaushik A., Langsley G, & Pain A. (2020). Novel tumour suppressor roles for GZMA and RASGRP1 in Theileria annulata‐transformed macrophages and human B lymphoma cells. Cellular Microbiology, 22(12), e13255.
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10

Fluorescent Labeling of MSC and Muscle-Derived Cells

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For the assessment of interactions in a direct co-culture, MSC were transfected with pMAX-GFP plasmid (Amaxa Biosystems) using the Nucleofector system (Amaxa Biosystems). Typically, 2x106 cells were in 100 μl Cell Line Nucleofector Human MSC Solution and mixed with 2 μg plasmid DNA. Nucleofections were performed using the Amaxa Nucleofector II device with a U-023 program. Post-nucleofection cells were incubated overnight in a RPMI medium supplemented with 20% FBS and used for the following procedures. In one experiment, muscle-derived cells were labeled with red membrane fluorochrome PKH26, and MSC were labeled with green PKH67 (both from Sigma Aldrich). Staining was performed according to the manufacturer instructions with a 4 nM of PKH solution for 4 min in RT. The labeling efficiency was evaluated with fluorescence microscopy (Olympus IX51) and is demonstrated in the supplementary material (S1 Fig).
Kulesza A., Burdzinska A., Szczepanska I., Zarychta-Wisniewska W., Pajak B., Bojarczuk K., Dybowski B, & Paczek L. (2016). The Mutual Interactions between Mesenchymal Stem Cells and Myoblasts in an Autologous Co-Culture Model. PLoS ONE, 11(8), e0161693.
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