Neon device

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Neon device is a laboratory equipment designed for various scientific applications. It serves as a platform for carrying out various analytical and experimental procedures in a controlled environment. The core function of the Neon device is to provide a stable and regulated environment for conducting research and analysis.

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Lab products found in correlation

Mabselect prisma resin, by Cytiva (1 mentions) 96 microplate luminometer, by Promega (1 mentions) Dual luciferase reporter gene assay kit, by Promega (1 mentions) Flt3l, by Thermo Fisher Scientific (1 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Neon transfection system, by Thermo Fisher Scientific (1 mentions) Stemspan sfem, by STEMCELL (1 mentions) U6 grna cmv cas9 gfp, by Merck Group (1 mentions)

Close spelling variants of this product

Neon transfection device Neon Transfection System device Neon Electroporation Transfection device Neon electroporation device Neon transfection kit and device NeonTM

6 protocols using neon device

Antibody Production in CHO Cells

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The variable light-chain and heavy-chain sequences were recovered from the 1F10 hybridoma and cloned into expression vectors in frame with constant regions to obtain a mouse IgG1/κ antibody harboring the N297Q mutation to abrogate binding to Fcγ receptors. The linearized vectors for the light chain and for the heavy chain were co-transfected into a CHO cell line using the Neon device (Invitrogen). The antibody was purified from the supernatant using the MabSelect PrismA resin (Cytiva). The antibody was eluted with citrate 0.1 M pH4.5 buffer, dialyzed overnight against PBS and 0.22 µM filtered. Size exclusion chromatography, SDS-PAGE and endotoxin levels quality controls were performed.

Tata A., Dodard G., Fugère C., Leget C., Ors M., Rossi B., Vivier E, & Brossay L. (2021). Combination blockade of KLRG1 and PD-1 promotes immune control of local and disseminated cancers. Oncoimmunology, 10(1), 1933808.

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Transient Transfection of Activated CD4+ T Cells

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The activated CD4⁺ T cells (2×10⁷ cells) were transfected transiently with pGPU6-shTIRC7/FLAG-CTLA-4 (10 µg/well) by electroporation methods using a Neon™ device according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were cultured in RPMI-1640 medium; expression of the plasmid was selected for using G418 (500 µg/ml; Gibco; Thermo Fisher Scientific, Inc.). After culture for 48 h, RNA and protein were collected and monitored by reverse transcription-quantitative PCR (RT-qPCR) and western blotting.

Zhu F., Qiu T., Zhu S., Zhao K., Chen C., Qiao J., Pan B., Yan Z., Chen W., Liu Q., Wu Q., Cao J., Sang W., Zeng L., Sun H., Li Z, & Xu K. (2020). TIRC7 inhibits Th1 cells by upregulating the expression of CTLA-4 and STAT3 in mice with acute graft-versus-host disease. Oncology Reports, 44(1), 43-54.

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STAT3 Luciferase Reporter Assay

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The luciferase reporter gene pGL-3-STAT3-luciferase (GLSTAT3-Lu) was synthesized by Beijing Yuan Ping Hao Biotechnology Co., Ltd. pGL-3-Basic was used in the control group. After GLSTAT3-Lu/pGL-3-Basic (10 µg/well) and FLAG-CTLA-4/FLAG (10 µg/well) were transfected into CD4⁺ T cells (2×10⁷ cells) by electroporation methods using a Neon™ device according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h, luciferase reporter gene activity was assessed using a dual-luciferase reporter gene assay kit, according to the manufacturer's protocol (Promega Corporation). The luciferase detection device used was a 96-microplate luminometer (Promega Corporation) and secreted alkaline phosphatase (cat. no. KA1362; Abnova) was used to normalize luciferase activity. Each experiment was repeated three times. STAT3 and phosphorylated (p)STAT3 protein levels were also monitored via western blotting. Anti-STAT3 (1:5,000; cat. no. ab119352) and anti-pSTAT3 (1:10,000; cat. no. ab76315) were both obtained from Abcam.

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Multiplexed CRISPR-Cas9 Genome Editing

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For electroporation, the gRNA complex was prepared according to the manufacturer’s recommendations by mixing Alt-R CRISPR–Cas9 crRNA (1 µM) and Alt-R transactivating crRNA (tracrRNA) coupled to ATTO 550 (IDT Inc.) to a final duplex concentration of 44 µM. The mixture was heated to 95 °C for 5 min and cooled down to room temperature for at least 60 min. For electroporation, 1.2 million Cas9 and reporter co-expressing cells were re-suspended in 120 µl R buffer and 4.9 µl of 44 µM gRNA mixture were added. 100 µl were subjected to each electroporation using the Neon device (Invitrogen, Eugene, OR, USA) under optimized conditions (1350 V, 10 ms, 3 pulses).
5 days after transient transfection of the LYN gRNA, single cells were sorted each in one well of a 96-well plate by FACSAria III according to GFP expression. Cells were allowed to expand for 2–3 weeks before they were transferred to 12-well plates and used for further analysis.

Liu W.H., Völse K., Senft D, & Jeremias I. (2021). A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models. Scientific Reports, 11, 12649.

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Genome Editing of CD34+ Stem Cells

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Human cord blood CD34⁺ stem cells were purchased from Lonza (Catalog #2C-101). The cells were thawed and cultured in stem cell maintenance media [StemSpan SFEM (STEMCELL Technologies), supplied with IL6 (50 ng/mL; Peprotech), TPO (50 ng/mL; Peprotech), SCF (50 ng/mL; Peprotech), and Flt-3L (50 ng/mL; Peprotech)]. After 2 days of culture, the cells were subjected to RNP Cas9/crRNA delivery. The crRNAs for knockout are listed in Supplementary Table S1. Briefly, the custom crRNA was synthesized using the Alt-R platform (IDT Technologies). Note that 5 μL of 100 μmol/L crRNA was annealed with 5 μL of 100 μmol/L tracrRNA and assembled with 6.5 μL of recombinant Cas9 protein (2.5 mg/mL). The mixture was added to 1 × 10⁶ CD34⁺ stem cells. The transfection was performed using the Neon Transfection System (Thermo Scientific; Catalog #MPK10025) and Neon device (Thermo Fisher). Specifically, the cells were resuspended in buffer T (as provided in the kit), and the electroporation was programmed to 1,600 V, 10 ms, and 3 pulses. After Cas9/crRNA delivery, the cells were recovered in the stem cell maintenance media for 3 days, followed by knockout validation by flow cytometry or Western blot. The antibodies used for knockout validation are listed in Supplementary Table S2.

Liu S.Q., Grantham A., Landry C., Granda B., Chopra R., Chakravarthy S., Deutsch S., Vogel M., Russo K., Seiss K., Tschantz W.R., Rejtar T., Ruddy D.A., Hu T., Aardalen K., Wagner J.P., Dranoff G, & D’Alessio J.A. (2020). A CRISPR Screen Reveals Resistance Mechanisms to CD3-Bispecific Antibody Therapy. Cancer immunology research, 9(1), 34-49.

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CRISPR/Cas9 Electroporation of Myoblasts

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5 × 10⁵DUPmyo (or DUPmyo-i) myoblasts were electroporated with 1 μg of the highly pure CRISPR/Cas9-GFP plasmids by using the NEON device (Thermo Fisher Scientific) provided by Julie Dumonceaux and Virginie Mariot, as specified by the manufacturer instructions. Number of pulses, duration and voltage intensity were suggested by Dumonceaux and Mariot, as follows: 1 pulse, 20 ms, 1400 V. Electroporated plasmids were U6gRNA-CMVCas9-GFP (Sigma-Aldrich) expressing sgRNA0 and sgRNA2 (named CR0 and CR2).

Pini V., Mariot V., Dumonceaux J., Counsell J., O’Neill H.C., Farmer S., Conti F, & Muntoni F. (2022). Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications. Scientific Reports, 12, 3756.

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