Neon electroporator

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Neon electroporator is a compact, versatile instrument designed for efficient and reliable DNA, RNA, or protein delivery into a variety of cell types. It utilizes electroporation technology to transfect cells with the desired molecular cargo. The Neon electroporator is capable of processing up to 16 samples simultaneously, providing a streamlined workflow for high-throughput applications.

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Lab products found in correlation

59 protocols using neon electroporator

Inducible Protein Expression and Nuclear Localization

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Hek 293T cells were
grown in DMEM with 10% FBS and plated on 35 mm dishes (MatTek corporation).
Transfection was performed with Lipofectamine (Invitrogen) according
to manufacturer’s instructions using 4 μg of each plasmid
required for the specific experiment. After transfection, caged lysine
was added yielding a final concentration of 2 mM. Cells were incubated
for 24 h until imaging. For the TEV cleavage experiment, MCF10A cells
stably selected for the Cumate inducible expression system (System
Biosciences) were grown in growth medium^{32 (link)} and transfected using the neon electroporator (Invitrogen) with
3 μg of each plasmid. Cumate was added for the first 10 h to
allow for SATB1 expression. After ∼10 h, caged lysine was added
yielding a final concentration of 2 mM to allow for TEV expression
and Cumate was removed to stop SATB1 expression and avoid premature
cleavage in the cytoplasm before import into the nucleus. Cells were
then incubated for another ∼6 h. Before imaging, in all experiments,
medium was replaced with DMEM containing no caged lysine. Nuclear
import of the FOXO3 transcription factor was monitored with the Cignal
FOXO GFP Reporter system (CCS-6022, www.sabiosciences.com), which consists of the GFP gene under the control of a minimal
(m)CMV promoter and tandem repeats of the FOXO transcriptional response
element.

Engelke H., Chou C., Uprety R., Jess P, & Deiters A. (2014). Control of Protein Function through Optochemical Translocation. ACS Synthetic Biology, 3(10), 731-736.

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Knockdown of SLC transporters in hASCs

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Studies were performed in hASCs cultured as described above. Cells were transfected using a Neon electroporator (Invitrogen, Carlsbad, CA) at day 4 of differentiation according to the manufacturer’s protocol. In brief, 1 million cells were mixed with 40 nmol/l ON-TARGETplus SMARTpool small interfering RNAs (siRNAs) targeting either SLC22A3, SLC29A4 or SLC47A1 or a non-targeting siRNA pool (Dharmacon, Lafayette, CO) and electroporated using a 100 μl NEON electroporation tip. Electroporation conditions were 1600 Volts, 20 ms width, 1 pulse. Subsequently, cells were plated in antibiotic-free medium at a density of 60.000 cells/well in 48-well plates. Medium was replaced 24 h post-transfection and subsequently every second day. The cells were cultured until day 12 of differentiation upon which lipolysis experiments were performed and protein/RNA/medium was collected. Apart from assessing cell viability by cell morphology, protein concentrations (in total cell lysates) or gene expression (e.g. of 18S rRNA), determination of lactate dehydrogenase activity (a measure of cell membrane integrity) was also performed in the conditioned media using Cytotoxicity Detection Kit (Roche). This showed no significant effect of our treatments (data not shown).

Arner P., Kulyté A., Batchelor K., Laurencikiene J., Livingston J, & Rydén M. (2018). MAPPING OF BIGUANIDE TRANSPORTERS IN HUMAN FAT CELLS AND THEIR IMPACT ON LIPOLYSIS. Diabetes, obesity & metabolism, 20(10), 2416-2425.

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Culturing Glioma Cells and Human Glioma Progenitor Cells

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C6 rat glioma cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in high-glucose DMEM supplemented with 10% heat-inactivated fetal bovine serum and glutamine (Invitrogen, Carlsbad, CA). Graded brain tumor specimens were obtained with informed consent as part of a study protocol approved by the SingHealth Centralised Institutional Review Board A. hGPCs were isolated and cultured as described previously (Chong et al., 2009 (link)) as tumor spheres in high-glucose DMEM/F-12 (3:1) supplemented with sodium pyruvate, nonessential amino acids, penicillin/streptomycin, B27 supplement (Invitrogen), basic fibroblast growth factor (bFGF; 20 ng/ml), epidermal growth factor (EGF; 20 ng/ml; Gene-Ethics (Asia), Singapore), and heparin (5 μg/ml; Sigma-Aldrich, St. Louis, MO). For transfection and migration assays, hGPCs were cultured as monolayers on laminin (10 μg/ml)–coated Petri dishes for several days before transfection. HGPCs and C6 transfections were performed with a Neon electroporator (Invitrogen) as per manufacturer's recommendations.

Monzo P., Chong Y.K., Guetta-Terrier C., Krishnasamy A., Sathe S.R., Yim E.K., Ng W.H., Ang B.T., Tang C., Ladoux B., Gauthier N.C, & Sheetz M.P. (2016). Mechanical confinement triggers glioma linear migration dependent on formin FHOD3. Molecular Biology of the Cell, 27(8), 1246-1261.

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Jurkat T Cell Transfection Protocol

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Jurkat E6.1 (CD4⁺) T cells were a kind gift from the Samelson lab at the NIH and Jurkat J76 (CD8⁺) cells were a kind gift from the Acuto lab at Oxford. These cells were transfected with DNA using a NEON electroporator (Invitrogen) for the expression of proteins tagged with PAFPs. Lines of Jurkat E6.1 cells, stably expressing TCRζ–Dronpa, were available for this study from previous work^{7 (link)}. Briefly, in that work the cells were created by selection with Geneticin at 1.5 mg ml⁻¹ (G418, Invitrogen). After 2–3 weeks, the cells were sorted and single clones were grown in 96 well plates. After 3 additional weeks, the extent of protein expression was checked by flow cytometry. Cells were then evaluated using biochemistry assays, flow cytometry, confocal microscopy (510 LSCM, Zeiss) and epifluorescence, TIRF and PALM imaging, as described below.

Razvag Y., Neve-Oz Y., Sajman J., Reches M, & Sherman E. (2018). Nanoscale kinetic segregation of TCR and CD45 in engaged microvilli facilitates early T cell activation. Nature Communications, 9, 732.

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Efficient Reprogramming of Oxygen-Sensitive iPSCs

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iPSCs were generated using transposon-mediated gene transfer (Woltjen et al., 2009, 2011 ). The transfections were done using Neon electroporator (Invitrogen; one pulse; 30 mS; 1,300 V) with 5 μg of DNA and 5 × 10⁵ cells per electroporation. The culture media was supplemented with 1 μg/ml of doxycycline on the next day. For reprogramming in different oxygen tensions, the electroporations were split on two separate plates: one cultured in 4% O₂ (Biospherix ExVivo chamber) whereas the other in normal room air of around 20% O₂. To assess the reprogramming efficiency, plates were stained on day 14 for alkaline phosphatase (AP) activity (Vector Red Alkaline Phosphate substrate kit). For clonality assays, cells were plated on day 0 on clonal dilutions and stained on day 7. The number of AP-positive clones was counted using ImageJ 1.44o software (NIH).
The screening for transgene silencing was done by lacZ staining (Woltjen et al., 2009, 2011 ), and karyotyping was done commercially (Chrombios). The iPSC growth curves were done by plating 2 × 10⁴ cells per well and counting the cell number on 4 consecutive days.

Hämäläinen R.H., Ahlqvist K.J., Ellonen P., Lepistö M., Logan A., Otonkoski T., Murphy M.P, & Suomalainen A. (2015). mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling. Cell Reports, 11(10), 1614-1624.

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Targeted Silencing in AML Cells

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Primary AML specimens were transfected with siRNA constructs targeting NAMPT, HADH, or a non-targeting scrambled siRNA (Dharmacon) following established protocols (Brunetti et al., 2018 ). Specifically, 2×10⁵ cells were electroporated using the Neon electroporator (Invitrogen) in Buffer T: R 1600, V 10 ms, 3 pulses.

Jones C.L., Stevens B.M., Pollyea D.A., Culp-Hill R., Reisz J.A., Nemkov T., Gehrke S., Gamboni F., Krug A., Winters A., Pei S., Gustafson A., Ye H., Inguva A., Amaya M., Minhajuddin M., Abbott D., Becker M.W., DeGregori J., Smith C., D'Alessandro A, & Jordan C.T. (2020). Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells. Cell stem cell, 27(5), 748-764.e4.

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Culturing and Transfecting Jurkat T Cells

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Jurkat (human leukemic) E6.1 (CD4⁺) T cells were a kind gift from the Samelson lab at the NIH. Jurkat cells were maintained in RPMI-1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2% glutamine, 2% sodium pyruvate and 2% HEPES. Cells were maintained in completely humidified air with 5% CO₂ at 37 °C. GFP-Fibrillarin Plasmid DNA (AddGene, UK), which was used in transfections, was purified from bacterial cultures using Maxiprep columns (QIAGEN, USA). Cells were transfected with the desired DNA plasmid by using a NEON electroporator (Invitrogen).

Wohl I, & Sherman E. (2019). ATP-Dependent Diffusion Entropy and Homogeneity in Living Cells. Entropy, 21(10), 962.

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Adipocyte Transfection Assay for Epigenetic Study

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Human mesenchymal stem cells (hMSCs) isolated from adipose tissue and differentiated in vitro to adipocytes were used for the transfection with plasmids^{42 (link)}. Day 9 of differentiated cells were transfected using Neon electroporator (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The cell amount per 10 µl electroporation tip was 100.000 together with 500 ng of methylated or unmethylated pCpGL-basic or pCpGL-PLIN plasmids and 10 ng of a plasmid containing Renilla luciferase gene (Promega, Madison, WI). Electroporation conditions were 1400 Volts, 20 ms width, and 2 pulses. Following electroporation the cells were cultured in 48-well plates for 24 hours there after luciferase activities were measured in cell lysates using Dual Luciferase Reporter Assay System (Promega) according manufacturer’s instructions. Each sample was prepared in quadruplicates and the experiment was repeated three times.

Bialesova L., Kulyté A., Petrus P., Sinha I., Laurencikiene J., Zhao C., Wright K.D., Arner P, & Dahlman I. (2017). Epigenetic Regulation of PLIN1 in Obese Women and its Relation to Lipolysis. Scientific Reports, 7, 10152.

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Analyzing DPAGT1 Exon Variants

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DPAGT1 exons 2, 3 and 4 and flanking intronic sequences or exons 6, 7 and 8 and flanking sequences were cloned into the pET01 vector (MoBiTec). c.478G>A and c.791T>G were respectively introduced by site-directed mutagenesis using Quikchange kit from Stratagene and confirmed by Sanger sequencing. Control and mutant vector DNA were electroporated into the human rhabdomyosarcoma cell line TE671 using the NEON electroporator (Invitrogen). Total RNA was purified 48 hr after transfection, reverse transcribed into cDNA using Retroscript kit (Ambion). cDNA was amplified using primers specific to the vector exons. The amplicons were run on agarose/TBE gels, visualized under UV/ethidium bromide and then gel purified and sequenced.

Dong Y.Y., Wang H., Pike A.C., Cochrane S.A., Hamedzadeh S., Wyszyński F.J., Bushell S.R., Royer S.F., Widdick D.A., Sajid A., Boshoff H.I., Park Y., Lucas R., Liu W.M., Lee S.S., Machida T., Minall L., Mehmood S., Belaya K., Liu W.W., Chu A., Shrestha L., Mukhopadhyay S.M., Strain-Damerell C., Chalk R., Burgess-Brown N.A., Bibb M.J., Barry III C.E., Robinson C.V., Beeson D., Davis B.G, & Carpenter E.P. (2018). Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design. Cell, 175(4), 1045-1058.e16.

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CRISPR-Cas9 Electroporation of FRDA Lymphoblasts

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Human FRDA lymphoblasts from the Coriell Institute (Table S2) were cultured in RPMI 1640/15% FBS at 37°C, 5% CO_2. 1 × 105 lymphoblasts were resuspended in 9 μL of electroporation buffer (buffer R, Invitrogen, Carlsbad, CA, USA); 1 μL of RNP complex was added as well as 1.8 μM Alt-R Cas9 electroporation enhancer (IDT, San Diego, CA, USA) when required. 10 μL was then pipetted into the 10-μL Neon tip (Invitrogen, Carlsbad, CA, USA), and the cell/RNP complex mixture was electroporated at 1,600 V, 10 ms, for three pulses using the Neon electroporator (Invitrogen, Carlsbad, CA, USA). Cells were immediately returned to pre-equilibrated cultured media in 24-well plates. The same protocol and number of cells were used for CD34⁺ HSPCs for in vitro experiments while 1 × 10⁶ CD34⁺ HSPCs (resuspended in 90 μL of buffer R) were electroporated with 10 μL of RNP complex for transplantation in one NSG mouse.

Rocca C.J., Rainaldi J.N., Sharma J., Shi Y., Haquang J.H., Luebeck J., Mali P, & Cherqui S. (2020). CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells from Patients with Friedreich’s Ataxia. Molecular Therapy. Methods & Clinical Development, 17, 1026-1036.

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