Amaxa nucleofector 96 well shuttle system

Manufactured by Lonza

The Amaxa Nucleofector 96-well Shuttle System is a laboratory instrument designed for high-throughput nucleofection, a method of delivering nucleic acids into mammalian cells. The system enables the efficient transfection of a large number of samples simultaneously, making it a versatile tool for various cell-based research applications.

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Lab products found in correlation

Nucleocuvette, by Lonza (2 mentions) 24 well plate, by BD (1 mentions) Poly d lysine, by Merck Group (1 mentions) Nucleocuvette plate, by Lonza (1 mentions) Nucleofector kit, by Lonza (1 mentions) Cas9 protein, by Thermo Fisher Scientific (1 mentions) Trilencer 27 human sirna, by OriGene (1 mentions) Silentfect lipid, by Bio-Rad (1 mentions) Human amphiregulin duoset elisa, by R&D Systems (1 mentions) P3 primary cells nucleofection kit, by Lonza (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Alexa 568, by Thermo Fisher Scientific (1 mentions) Fc010, by Merck Group (1 mentions)

6 protocols using amaxa nucleofector 96 well shuttle system

Primary Rat Cortical Neuron Transfection

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Postnatal day 3 Sprague-Dawley rat pups (Charles River) were sacrificed and their cortices dissected as described in (Mehta et al., 2016 (link)). 150,000 cells were then resuspended in 20μL of P3 Primary Cell Nucleofector Solution + Supplement (Lonza; Catalog #V4SP-3096) with 1.7μg of plasmid DNA and transfected using the AMAXA Nucleofector 96-well Shuttle System (program: Neurons, High Viability; Lonza). Cells were then plated at a concentration of 15,000 cells per well in 24-well plates (Falcon) coated with 0.5mg/mL poly-D-lysine (Sigma). Three technical replicate wells were used per condition per biological replicate. Cells were cultured in Proneural Growth Medium supplemented with PNGM SingleQuots (Lonza; Catalog # CC4461) conditioned overnight using cultured P5 rat glia. Cortical neurons were grown for 2 days in culture at 37°C with 5% CO2 before fixation and immunofluorescent staining. The bottom row of each plate contained non-transfected cells in each well as a negative control for the staining.

Danzi M.C., Mehta S.T., Dulla K., Zunino G., Cooper D.J., Bixby J.L, & Lemmon V.P. (2018). The effect of Jun dimerization on neurite outgrowth and motif binding. Molecular and cellular neurosciences, 92, 114-127.

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Base Editing in K562 Cells

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K562 cells (ATCC, CCL243) were cultured using RPMI-1640 growth medium supplemented with 10% FBS (Fetal Bovine Serum) and 1x PSG (Penicillin-Streptomycin-Gentamycin, Gibco, 10378-016) and maintained at 37 °C with 5% CO₂. ZF-CBEs and GFP were dosed as plasmid DNA (pDNA) in K562 cells. K562 cells were electroporated with pDNA using the SF cell line 96-well Nucleofector kit (Lonza, V4SC-2960) using manufacturer’s protocol. Prior to electroporation, K562 cells were centrifuged at ~300 × g for 5 min. Cells were resuspended at 2e5 cells per 12 µl of supplemented SF cell line 96-well Nucleofector solution. Twelve µl of cells were mixed with 8 µl of pDNA (400 ng of each ZF-CBE-L, ZF-CBE-R and optionally the nickase construct) and transferred to the Lonza Nucleocuvette plate. Nucleofector program 96-FF-120 was used to electroporate K562 cells with the pDNA mix on the Amaxa Nucleofector 96-well Shuttle System (Lonza). After electroporation, cells were incubated for 10 min at room temperature and transferred to a 96-well tissue culture plate containing 180 µl of complete medium (prewarmed to 37 °C). K562 cells were incubated for ~72 h and then harvested for base editing and indel quantification.

Fauser F., Kadam B.N., Arangundy-Franklin S., Davis J.E., Vaidya V., Schmidt N.J., Lew G., Xia D.F., Mureli R., Ng C., Zhou Y., Scarlott N.A., Eshleman J., Bendaña Y.R., Shivak D.A., Reik A., Li P., Davis G.D, & Miller J.C. (2024). Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells. Nature Communications, 15, 1181.

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Efficient CRISPR-Cas9 Delivery in Cells

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Cells were resuspended in high-efficiency buffer, with a crRNA and tracrRNA (Integrated DNA Technologies) complex and Cas9 protein (ThermoFisher). Cells were electroporated in nucleocuvettes (Lonza) using program EH-100 on the Amaxa Nucleofector 96-well Shuttle System (Lonza), and transferred to prewarmed complete medium.

Kubo S., Fritz J.M., Raquer-McKay H.M., Kataria R., Vujkovic-Cvijin I., Al-Shaibi A., Yao Y., Zheng L., Zou J., Waldman A.D., Jing X., Farley T.K., Park A.Y., Oler A.J., Charles A.K., Makhlouf M., AbouMoussa E.H., Hasnah R., Saraiva L.R., Ganesan S., Al-Subaiey A.A., Matthews H., Flano E., Lee H.H., Freeman A.F., Sefer A.P., Sayar E., Çakır E., Karakoc-Aydiner E., Baris S., Belkaid Y., Ozen A., Lo B, & Lenardo M.J. (2021). Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease. Nature immunology, 23(1), 75-85.

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Silencing AREG Secretion in A549 Cells

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T cells were resuspended in high-efficiency buffer (140 mM sodium phosphate (pH 7.2), 5 mM KCl and 10 mM MgCl₂) with siRNA (Trilencer-27 Human siRNA, Origene). Cells were transferred to Nucleocuvettes (Lonza) and electroporated using program EO-115 on the Amaxa Nucleofector 96-well Shuttle System (Lonza). After electroporation, cells were transferred to prewarmed complete medium.
A549 cells (1 × 10⁵ cells per well) were seeded in a 12-well plate containing complete medium and transfected with siRNA using siLentfect Lipid (BioRad) reagent. To quantify AREG secretion, medium was replaced 48 h following lipofection and collected 24 h later, for analysis by the Human Amphiregulin DuoSet ELISA (R&D Systems).

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Nucleofection of GM01953 and Primary Lymphocytes

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Both GM01953 cells and primary lymphocytes were transfected with the P3 primary cells nucleofection kit (Lonza) using the 96-E0-115 program recommended for “high functionality” transfection of uninduced T-cells on the Amaxa Nucleofector 96-well Shuttle system. Typical transfections used 150,000–190,000 GM01953 or 125,000–200,000 primary lymphocytes (plus a variable amount of other WBCs depending on the sample preparation method). One individual aliquot of frozen GM01953 or primary lymphocytes was used for each experimental day to generate 3 measurements of each type of repair (NHEJ and SSA) plus all controls for the FACS analysis (1–3 measurements depending on the experimental day for GM01953).

Lacoste S., Bhatia R., Bhatia S, & O'Connor T.R. (2014). Granulocytes Affect Double-Strand Break Repair Assays in Primary Human Lymphocytes. PLoS ONE, 9(3), e93185.

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Multicolor Live-cell Imaging of Clathrin-mediated Endocytosis

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BSC-1, COS-7, U2OS, and mouse embryonic fibroblast (MEF) cells (American Type Culture Collection) were grown to ~60 to 80% confluency in Dulbecco’s modified eagle medium (DMEM) with high glucose and no phenol red supplemented with 15% fetal bovine serum (Life Technologies). BSC-1 cells stably expressed EGFP-CLTA. Other cells were transiently transfected with an Amaxa Nucleofector 96-well shuttle system (Lonza) with 1 μg DNA per 400,000 cells with nucleofection solution and a program optimized for each cell line per the manufactures instructions. Before imaging, 25-mm or 5-mm coverslips were coated with 10 μg/ml fibronection (Millipore, FC010) for 24 hours before plating transfected cells. Imaging was performed in DMEM with HEPES if there is no CO₂ control containing no phenol red at temperatures specifically stated in each case.
In two-color imaging of CCPs and transferrin receptors (TfRs) by means of high-NA TIRF-SIM, MEF cells expressing clathrin light chain B fused to the C terminal of mEmerald were incubated with DMEM medium containing 250 μg/mL TfR bound to human transferrin conjugated with Alexa 568 (T23365, Life Technologies) for 15 min.
Fixed cells were treated for 15 min with fixation buffer containing 4% paraformaldehyde, 0.1% gluteraldehyde in PHEM buffer (25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, and 120 mM PIPES in pH 7.3).

Li D., Shao L., Chen B.C., Zhang X., Zhang M., Moses B., Milkie D.E., Beach J.R., Hammer JA I.I.I., Pasham M., Kirchhausen T., Baird M.A., Davidson M.W., Xu P, & Betzig E. (2015). Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science (New York, N.Y.), 349(6251), aab3500.

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