Neon transfection system mpk5000

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Neon Transfection System MPK5000 is a laboratory instrument designed for the efficient delivery of nucleic acids, such as DNA and RNA, into a variety of cell types. It utilizes electroporation technology to facilitate the uptake of the transfection material by the cells.

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Neon Transfection System Neon transfection MPK5000 Neon system NeonTM

12 protocols using neon transfection system mpk5000

Transfection of STAT1 plasmids in cell lines

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HL-60, HL-60/Adr (Adriamycin resistant) and U937 (Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China) cells were cultured in RPMI 1640. HeLa cells (American Type Culture Collection, Rockville, MD, USA) were grown in DMEM. Primary leukemic cells from AML patients without prior therapy (the First Affiliated Hospital of Nanjing Medical University, Nanjing, China) were collected using lymphocyte-monocyte separation medium (KeyGen BioTECH, Nanjing, China), after obtaining informed consent, in agreement with the hospital’s Institutional Review Board and in accordance with the Declaration of Helsinki. The procedures were approved by the appropriate ethics committees.
EGFP-STAT1-WT plasmids, EGFP-STAT1 mutants (S727A, S727E, Y701F) and EGFP-pcDNA3.1 vectors were obtained from Addgene (Cambridge, MA, USA). HL-60 cells were electrotransfected with plasmids using Neon Transfection System MPK5000 (Invitrogen, Carlsbad, CA, USA). HeLa cells were transfected using Lipofectamine 3000 (Invitrogen).

Gao J., Fan M., Xiang G., Wang J., Zhang X., Guo W., Wu X., Sun Y., Gu Y., Ge H., Tan R., Qiu H., Shen Y, & Xu Q. (2017). Diptoindonesin G promotes ERK-mediated nuclear translocation of p-STAT1 (Ser727) and cell differentiation in AML cells. Cell Death & Disease, 8(5), e2765-.

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Silencing TβRII in NB4 Leukemia Cells

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NB4 cells were cultured in RPMI 1640 medium (Gibco/BRL) supplemented with 10% FBS. Lentiviral vectors designed to express human TβRII siRNA or TβRII splice variants (Mu), and negative control vectors were constructed with the aid of Shanghai GeneChem. The siRNA sequences targeting TβRII were designed to silence both endogenous TβRII splice variants using an Invitrogen Neon Transfection System (MPK5000). A double-stranded oligonucleotide encoding the shRNA sequence, a mismatched siRNA mutant, and sequences encoding the two TβRII splice variants (Mu) tagged with green fluorescent protein (GFP) were annealed and inserted into the pGC-LV expression vector (Invitrogen). The siRNA and TβRII splice variant (Mu) expression vectors (pGC-LV), and the packaging vectors (pHelper 1.0 and pHelper 2.0; Invitrogen) were cotransfected into 293FT cells using Lipofectamine 2000 (Invitrogen). The culture supernatants were collected, concentrated, and stored at -70°C. Enhanced GFP (eGFP) was expressed in all lentiviral vectors to measure viral titer and infection efficiency. The lentiviral vectors were introduced into NB4 cells in the presence of polybrene (10 mg/mL), with an MOI of 5–20.

Wu Y., Su M., Zhang S., Cheng Y., Liao X.Y., Lin B.Y, & Chen Y.Z. (2016). Abnormal expression of TGF-beta type II receptor isoforms contributes to acute myeloid leukemia. Oncotarget, 8(6), 10037-10049.

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CRISPR Knockin Screening Protocol

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Guide‐it Complete sgRNA Screening Systems (632 636; Clontech, CA, USA) were used for the in vitro transcription and efficiency testing of sgRNAs. Donor DNAs with mutant sites were produced using the Guide‐it Long ssDNA Production System (632 644; Clontech). sgRNAs, donor DNAs, together with Guide‐it Recombinant Cas9 (632 640; Clontech) were imported into cells via electroporation using the Neon Transfection System (MPK5000; Invitrogen, CA, USA). Cells were allowed to recover for 72 h after electroporation then seeded into 96‐well plates for single clone selection. After primary selection by the Guide‐it Knockin Screening Kit (632 660; Clontech), positive clones were further validated by DNA sequencing for successful mutation of targeted sites.

Luo Q., Wu X., Zhao P., Nan Y., Chang W., Zhu X., Su D, & Liu Z. (2021). OTUD1 Activates Caspase‐Independent and Caspase‐Dependent Apoptosis by Promoting AIF Nuclear Translocation and MCL1 Degradation. Advanced Science, 8(8), 2002874.

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Overexpression and Knockdown of EZH2 in PASMCs

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For EZH2 overexpression, the EZH2 gene was subcloned into the pEZ-M98 vector (GeneCopoeia, Inc.) to generate pM-EZH2. Cultured N-PASMCs were then transfected with pM-EZH2 vector using the Neon Transfection System (MPK5000) from Invitrogen (Thermo Fisher Scientific, Inc.), according to the supplier's instructions. Briefly, cells were cultured in 60-mm dishes to 70% confluency and then transfected with 2 µg plasmid DNA (1,375 V; 20 ms) at room temperature. Empty vector pEZ-M98 (pM) was used as a transfection control.
For EZH2 knockdown, PASMCs from CTEPH patient 3 were cultured in 60-mm dishes and transfected with 160 pmol small interfering (si)RNA (Shanghai GenePharma Co., Ltd.) and treated with Lipofectamine RNAiMAX reagent from Invitrogen (Thermo Fisher Scientific, Inc.) at room temperature, according to the manufacturer's instructions. The sequences were sense, 5′-CCUGACCUCUGUCUUACUUTT-3′ and antisense, 5′-AAGUAAGACAGAGGUCAGGTT-3′. RNAi negative control that had no homology with mammalian genes was used as the transfection control. The RNAi negative control sequences were sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. After 48 h, transfection efficiency was determined by both PCR and western blot analysis for EZH2.

Wang Y., Huang X.X., Leng D., Li J.F., Liang Y, & Jiang T. (2020). Effect of EZH2 on pulmonary artery smooth muscle cell migration in pulmonary hypertension. Molecular Medicine Reports, 23(2), 129.

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Fibroblast Electroporation with Base Editor

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The Neon™ Transfection System MPK5000 (Invitrogen) was used to electroporate the fibroblasts with the base editor and sgRNA. The base editing reagents (sgRNA and ABE) for each condition were prepared in a separate well of a 96-well plate and made up to a total volume of 10 μl with TE buffer. 1 × 10⁶ Fibroblasts were pelleted (1200 rpm, 5 min), and resuspended in 100 μl Resuspension Buffer R (Neon kit) per condition just before electroporation. 100 μl of the cell suspension was mixed with the base editing reagents, taken up in a 100 μl Neon™ tip ensuring there is no bubble at the top, placed in the pipette holder containing 3 ml of Buffer E2 (Neon kit) and electroporated at Voltage = 1500 V; Width = 20 ms; # pulses = 1 pulse.

Sheriff A., Guri I., Zebrowska P., Llopis-Hernandez V., Brooks I.R., Tekkela S., Subramaniam K., Gebrezgabher R., Naso G., Petrova A., Balon K., Onoufriadis A., Kujawa D., Kotulska M., Newby G., Łaczmański Ł., Liu D.R., McGrath J.A, & Jacków J. (2022). ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation. Scientific Reports, 12, 19643.

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Electroporation of KG1 Cells with STAT5 Plasmid

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Electroporation of plasmid DNA into KG1 cells was performed using Neon Transfection System MPK5000 (Invitrogen) and 100 µL NeonTip. Cells were plated in a complete medium at 0.6 × 10⁶/mL the day before electroporation. KG1 cells (5 × 10⁶) were electroporated with 10 µg of pMIG or pMIG-STAT51*6 plasmid (1700 V, 20 ms, 1 impulse).

Polak A., Bialopiotrowicz E., Krzymieniewska B., Wozniak J., Stojak M., Cybulska M., Kaniuga E., Mikula M., Jablonska E., Gorniak P., Noyszewska-Kania M., Szydlowski M., Piechna K., Piwocka K., Bugajski L., Lech-Maranda E., Barankiewicz J., Kolkowska-Lesniak A., Patkowska E., Glodkowska-Mrowka E., Baran N, & Juszczynski P. (2020). SYK inhibition targets acute myeloid leukemia stem cells by blocking their oxidative metabolism. Cell Death & Disease, 11(11), 956.

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FOXRED1 Knockout in HEK293T Cells

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HEK293T cells were electroporated (Neon Transfection System, MPK5000, ThermoFisher) with Alt-R Sp HiFi Cas9 nuclease v3, tracrRNA, and FOXRED1 crRNA (Hs.Cas9.FOXRED1.1.AA and Hs.Cas9.FOXRED1.1.AC), all from Integrated DNA Technologies (Coralville, IA) per the manufacturer’s instructions. Single clones were expanded and plated in both DMEM + 10% FBS media containing 0.4 mM uridine, 2 mM pyruvate and either 25 mM glucose or 10 mM galactose. Clones that failed to grow in galactose had genomic DNA Sanger sequenced for evidence of editing, and clones were subject to Western blot to confirm FOXRED1 deletion, and one of these that appeared to grow well was further studied.

Balderas E., Eberhardt D.R., Lee S., Pleinis J.M., Sommakia S., Balynas A.M., Yin X., Parker M.C., Maguire C.T., Cho S., Szulik M.W., Bakhtina A., Bia R.D., Friederich M.W., Locke T.M., Van Hove J.L., Drakos S.G., Sancak Y., Tristani-Firouzi M., Franklin S., Rodan A.R, & Chaudhuri D. (2022). Mitochondrial calcium uniporter stabilization preserves energetic homeostasis during Complex I impairment. Nature Communications, 13, 2769.

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CRISPR-Mediated Knockout of DND41 PE Cells

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DND41 PE^+/− cells were generated following a previously described protocol (59 (link)). Briefly, custom primers (Sigma-Aldrich) (Supplementary Table S3) were used to generate sgRNA DNA templates. Evaluation for potential off-targets was performed through CRISPRscan (https://www.crisprscan.org). In vitro transcription of sgRNAs from templates was performed following the protocol guidelines. 500ng of each purified sgRNA was independently incubated with 500ng of Cas9 (1074181, IDT) for 20 minutes at RT. Subsequently, one million DND41 cells were electroporated with 1μg of each complex per transfection using the Neon Transfection System (MPK5000, Thermo Fisher Scientific) and 100μL electroporating tips (MPK10025, Thermo Fisher Scientific). Electroporation conditions: pulse voltage 1350v, pulse width 10ms, pulse number 3, cell density 107/ml. Single cell clones were sorted using the Influx High Speed Sorter (BD Biosciences) and were subsequently grown in tissue culture and screened for PE loss by PCR using REDTaq ReadyMix (R2523–100RXN, Sigma Aldrich) following standard conditions and using custom primers (Supplementary Table S3).

Tottone L., Lancho O., Loh J.W., Singh A., Kimura S., Roels J., Kuchmiy A., Strubbe S., Lawlor M.A., da Silva-Diz V., Luo S., Gachet S., García-Prieto C.A., Hagelaar R., Esteller M., Meijerink J.P., Soulier J., Taghon T., Van Vlierberghe P., Mullighan C.G., Khiabanian H., Rocha P.P, & Herranz D. (2020). A Tumor Suppressor Enhancer of PTEN in T-cell Development and Leukemia. Blood Cancer Discovery, 2(1), 92-109.

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TCam-2 Cell Culture and Transfection

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TCam-2 [43 (link),44 (link),45 (link)] cells were cultured in RPMI (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% fetal calf serum (Thermo Fisher, Waltham, MA, USA). To avoid mycoplasma contamination cells were regularly tested with MycoAlert (Lonza, Basel, Switzerland). TCam-2 cells were transfected using the Neon Transfection System MPK5000 (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. For transfection of miRNA oligonucleotides, cells were transfected with 50 nmol of miRNA precursors or control non-targeting scrambled oligonucleotides. Transfection efficiency was verified for each experiment by evaluating GFP expression.

De Martino M., Esposito F., Pellecchia S., Cortez Cardoso Penha R., Botti G., Fusco A, & Chieffi P. (2020). HMGA1-Regulating microRNAs Let-7a and miR-26a are Downregulated in Human Seminomas. International Journal of Molecular Sciences, 21(8), 3014.

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Overexpression of Human MKP-3 in Cells

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To transiently overexpress MKP-3, a DNA fragment containing full-length human MKP-3 coding sequences (bp 1–1,146) was generated by PCR amplification of MKP-3 plasmid (a kind gift from Dr. Tae Jun Park, Ajou University, Suwon, Republic of Korea) using the forward primer 5′-GCTGGATATCATGATAGATACGCTCAGACC-3′ and reverse primer 5′-TCGAGCGGCCGCTCACGTAGATTGCAGAGAGT-3′, followed by cloning into EcoRV- and NotI-restricted multiple cloning sites of pcDNA3.1 (Invitrogen). DNA electroporation was performed using the Neon Transfection System MPK5000 (Thermo Fisher Scientific). Subsequently, 5 × 10⁵ cells were used per electroporation using Neon Transfection System 100 μL Kit (Thermo Fisher Scientific) under the following condition: one pulse of 1,350 V for 30 ms.

Unenkhuu B., Kim D.B, & Kim H.S. (2021). MKP-3 suppresses LPS-induced inflammatory responses in HUVECs via inhibition of p38 MAPK/NF-κB pathway. Animal Cells and Systems, 25(4), 235-244.

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