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Amaxa basic nucleofector kit for primary mammalian smooth muscle cells

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The Amaxa Basic Nucleofector Kit for Primary Mammalian smooth muscle cells is a laboratory equipment product designed for the transfection of primary mammalian smooth muscle cells. It is used for the introduction of nucleic acids, such as DNA or RNA, into these cell types.

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Lab products found in correlation

Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Rneasy kit, by Qiagen (1 mentions) Smad3 sirna, by Thermo Fisher Scientific (1 mentions) Cc 3182, by Lonza (1 mentions) Puromycin, by Merck Group (1 mentions)

Close spelling variants of this product

Amaxa Nucleofector (332 citations) Nucleofector kit (131 citations) Amaxa Nucleofector Kit (71 citations) Amaxa Basic Nucleofector Kit (24 citations) Basic Nucleofector Kit (11 citations) Amaxa basic nucleofector kit for primary (3 citations) Amaxa kits (2 citations)

5 protocols using amaxa basic nucleofector kit for primary mammalian smooth muscle cells

1

Silencing and Overexpressing SMAD3 in HCASMCs

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SMAD3 (s8401 and s8402) silencer select siRNAs were purchased from Life Technologies. siRNA transfection was performed using Lipofectamine RNAiMAX (Life Technologies). For each well treated with the SMAD3 siRNA or scrambled control (Life technologies, #4390843), the final concentration was 20 nM. HCASMCs were seeded in 6 well plates and grown to 75% confluence before siRNA transfection. HCASMCs were transfected with the SMAD3 siRNA or scrambled control for 12 hours and subsequently collected and processed for RNA isolation after 48 hrs of transfection using the RNeasy kit (Qiagen). For the SMAD3 overexpression study, HCASMCs were transduced with 5ug of pRK5F-SMAD3 cDNA (Addgene plasmid# 12625) or control pCDNA3.1 DNA (ThermoFisher Scientific, plasmid# V79020) using the Amaxa Basic Nucleofector kit for primary mammalian smooth muscle cells (Lonza #VPI-1004) at a density of 1x106 cells per 100 μL sample using Nucleofector Program U-025. Cells were changed to medium with supplements 24hrs after transfection and cultured for an additional 48 hrs. The transduction efficiency was assessed by transducing HCASMCs with 2 μg of pmax-GFP cDNA and quantifying the percentage of GFP positive cells by quantitative fluorescence microscopy.
Iyer D., Zhao Q., Wirka R., Naravane A., Nguyen T., Liu B., Nagao M., Cheng P., Miller C.L., Kim J.B., Pjanic M, & Quertermous T. (2018). Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk. PLoS Genetics, 14(10), e1007681.
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2

Transfection of HCASMC with TCF21 siRNA

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Human primary Coronary Artery Smooth Muscle Cells (HCASMC, Lonza #CC-2583 Lot 200212 and Cell Applications # 350-05a Lot 1508) were cultured in Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics (Lonza, #CC-3182). For RNA-Seq studies donor-pooled HCASMC were transfected with 300 nM TCF21 Trilencer-27 Human siRNA (OriGene #SR304753C) or Trilencer-27 Universal Scrambled Negative Control siRNA (OriGene #SR30004) at 80% confluence using the Amaxa Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells (Lonza #VPI-1004) at a density of 1 × 106 cells per 100 μL sample using Nucleofector Program U-025. Cells were changed to medium with supplements at 18 hours post-transfection and cultured for an additional 48 hours.
Nurnberg S.T., Cheng K., Raiesdana A., Kundu R., Miller C.L., Kim J.B., Arora K., Carcamo-Oribe I., Xiong Y., Tellakula N., Nanda V., Murthy N., Boisvert W.A., Hedin U., Perisic L., Aldi S., Maegdefessel L., Pjanic M., Owens G.K., Tallquist M.D, & Quertermous T. (2015). Coronary artery disease associated transcription factor TCF21 regulates smooth muscle precursor cells that contribute to the fibrous cap. Genomics Data, 5, 36-37.
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3

HOXA13 Overexpression in Smooth Muscle Cells

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HOXA13 overexpression plasmid, pLV(Exp)-EGFP:T2A:Puro-CBh > hHOXA13, vector ID VB180306-1076naw, was constructed and packaged by VectorBuilder (Cyagen Bioscience). Nucleofection was carried out using Amaxa Basic Nucleofector Kit for Primary Mammalian smooth muscle cells (Lonza, Catalog # VPI-1004) according to manufactures protocol. Briefly, 1X106 cells was resuspended in 100μl Nucleofector solution, and transfected with 1μg of HOXA13 overexpression plasmid using program P-024. Following nucleofection, cells were incubated for 18 hours and media was changed to complete growth media along with supplements. 48 hours after nucleofection, the cells were treated with 2μg/ml puromycin to select for stable expression. Expression analysis was determined by qRT-PCR on cloned cells (n=4) with RPL17 as the housekeeping gene.
George J.W., Fan H., Johnson B., Carpenter T.J., Foy K.K., Chatterjee A., Patterson A.L., Koeman J., Adams M., Madaj Z.B., Chesla D., Marsh E.E., Triche T.J., Shen H, & Teixeira J.M. (2019). Integrated Epigenome, Exome, and Transcriptome Analyses Reveal Molecular Subtypes and Homeotic Transformation in Uterine Fibroids. Cell reports, 29(12), 4069-4085.e6.
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4

MSC Stable Transfection with mCherry

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PiggyBac transposon‐based strategy was used for MSCs stable transfection with mCherry (Yusa, Zhou, Li, Bradley, & Craig, 2011). The coding region of mCherry was amplified by PCR and cloned into the XbaI/EcoRI sites of the backbone PiggyBac vector PB‐CMV‐MCS‐EF1‐Puro (System Biosciences; Cat. No. PB5105B‐1). For further genomic integration, the following vector was used: pCMV‐hyPBase (provided by the Wellcome Trust Sanger Institute). MSCs obtained from 2 and 20 months old male rats were transfected with PB‐CMV‐mCherry‐EF1‐Puro and pCMV‐hyPBase plasmids. Amaxa® Basic Nucleofector® Kit for Primary Mammalian Smooth Muscle Cells (Lonza) and the program U‐023 were used to transfect 5 × 106 cells with 4 μg of PB‐CMV‐mCherry‐EF1‐Puro and 1 μg of pCMV‐hyPBase. This methodology allowed approximately 40% of transfection efficiency. For further selection, transfected cells were incubated in the presence of 2 μg/mL puromycin (Sigma) for at least 1 week, leading to a highly pure mCherry‐expressing MSCs culture.
Rivera F.J., de la Fuente A.G., Zhao C., Silva M.E., Gonzalez G.A., Wodnar R., Feichtner M., Lange S., Errea O., Priglinger E., O'Sullivan A., Romanelli P., Jadasz J.J., Brachtl G., Greil R., Tempfer H., Traweger A., Bátiz L.F., Küry P., Couillard‐Despres S., Franklin R.J, & Aigner L. (2019). Aging restricts the ability of mesenchymal stem cells to promote the generation of oligodendrocytes during remyelination. Glia, 67(8), 1510-1525.
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5

HOXA13 Overexpression in Smooth Muscle Cells

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HOXA13 overexpression plasmid, pLV(Exp)-EGFP:T2A:Puro-CBh > hHOXA13, vector ID VB180306-1076naw, was constructed and packaged by VectorBuilder (Cyagen Bioscience). Nucleofection was carried out using Amaxa Basic Nucleofector Kit for Primary Mammalian smooth muscle cells (Lonza, Catalog # VPI-1004) according to manufactures protocol. Briefly, 1X106 cells was resuspended in 100μl Nucleofector solution, and transfected with 1μg of HOXA13 overexpression plasmid using program P-024. Following nucleofection, cells were incubated for 18 hours and media was changed to complete growth media along with supplements. 48 hours after nucleofection, the cells were treated with 2μg/ml puromycin to select for stable expression. Expression analysis was determined by qRT-PCR on cloned cells (n=4) with RPL17 as the housekeeping gene.
George J.W., Fan H., Johnson B., Carpenter T.J., Foy K.K., Chatterjee A., Patterson A.L., Koeman J., Adams M., Madaj Z.B., Chesla D., Marsh E.E., Triche T.J., Shen H, & Teixeira J.M. (2019). Integrated Epigenome, Exome, and Transcriptome Analyses Reveal Molecular Subtypes and Homeotic Transformation in Uterine Fibroids. Cell reports, 29(12), 4069-4085.e6.
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