Human t cell nucleofection kit

Manufactured by Lonza

Sourced in United States

The Human T-cell nucleofection kit is a laboratory equipment product designed to facilitate the transfection of genetic material into human T-cells. It provides the necessary components and protocols to efficiently introduce plasmid DNA, mRNA, or other nucleic acids into T-cells using the nucleofection technique.

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Lab products found in correlation

Amaxa nucleofection system, by Lonza (2 mentions) Non targeting shrna control, by Merck Group (1 mentions) Gene pulser, by Bio-Rad (1 mentions) Anti human cd3, by Thermo Fisher Scientific (1 mentions) Anti human cd3 fitc, by BD (1 mentions) Pe anti human cd123, by BD (1 mentions) Apc cy7 anti human hla dr, by BioLegend (1 mentions) Pan dc enrichment kit, by Miltenyi Biotec (1 mentions) Bv421 anti human cd11c, by BioLegend (1 mentions) Legendplex th cytokine panel, by BioLegend (1 mentions)

Spelling variants of this product

Nucleofection (130 citations) Nucleofection Kit (19 citations) Human nucleofection kit (4 citations)

5 protocols using human t cell nucleofection kit

Electroporation of CD3+ T Cells

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The electroporation of CD3+ cells was performed with the Amaxa Nucleofector system (Amaxa Human T cell Nucleofection Kit). For that the cells were resuspended in Nucleofector solution with 50 µg/ml of plasmid and treated with program U-14 according to the manufacturer’s instructions. Immediately after that cells were transferred into 500 µl prewarmed media and were additionally washed with fresh media before their culturing.

Lukianova-Hleb E.Y., Yvon E.S., Shpall E.J, & Lapotko D.O. (2016). All-in-one processing of heterogeneous human cell grafts for gene and cell therapy. Molecular Therapy. Methods & Clinical Development, 3, 16012-.

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PKCζ Knockdown in Human CB T-Cells

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PKCζ knockdown in human CB T-cells was carried out essentially as described recently for human macrophages [31 (link)]. Predesigned shRNA specific for PKCζ and non-targeting control shRNA were purchased from Sigma–Aldrich (Castle Hill, NSW, Australia). The human T-cell Nucleofection kit was from Amaxa (Lonza, Wakersville, MD, U.S.A.). Approximately 10⁶ cells were added to 4 μg of non-targeting control shRNA or PKCζ-specific shRNA in cuvettes and the cells were transfected using programme Y-010 according to the manufacturer’s instructions. After transfection, T-cells were cultured for 24 h before harvesting for maturation studies. An aliquot of the cultures was used to confirm the knockdown of PKCζ by Western blot analysis. Cell viability monitored by the Trypan Blue dye exclusion assay was >90%, being consistent with the information provided by the Nucleofection kit.

Harb H., Irvine J., Amarasekera M., Hii C.S., Kesper D.A., Ma Y., D’Vaz N., Renz H., Potaczek D.P., Prescott S.L, & Ferrante A. (2017). The role of PKCζ in cord blood T-cell maturation towards Th1 cytokine profile and its epigenetic regulation by fish oil. Bioscience Reports, 37(2), BSR20160485.

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Culturing and Transfecting Trypanosoma brucei

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Procyclic-form (PCF) Lister 427 Trypanosoma bruceibrucei, bloodstream-form (BSF) Lister 427 and single-marker bloodstream-form (SMB) parasites were cultured as previously described (Brun and Schönenberger, 1979 (link); Hirumi and Hirumi, 1989 (link); Wirtz et al., 1999 (link)). For generation and maintenance of lines harbouring selectable markers, antibiotics were used at the following concentrations: G418, 1 μg/ml; puromycin, 0.2 μg/ml; and hygromycin B, 5 μg/ml. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM L-glutamine. For transfection of bloodstream-stage parasites, 3×10⁷–4×10⁷ cells were transfected with 10 μg of DNA using an AMAXA nucleofection system and the human T-cell nucleofection kit (Lonza). Monoclonal populations were obtained by limiting dilution. Procyclic-stage parasites were transfected by electroporation with a Bio-Rad Gene Pulser (1.5 kV, 25 μF). COS-7 cells were transfected with Fugene HD according to manufacturer's instructions.

Manna P.T., Obado S.O., Boehm C., Gadelha C., Sali A., Chait B.T., Rout M.P, & Field M.C. (2017). Lineage-specific proteins essential for endocytosis in trypanosomes. Journal of Cell Science, 130(8), 1379-1392.

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Culturing and Transfecting Trypanosoma brucei

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Bloodstream-form Trypanosoma brucei strain Lister 427 parasites were cultured in HMI-9 medium supplemented with 10% fetal bovine serum (Hirumi and Hirumi, 1989 (link)). For RNAi experiments, the tetracycline-responsive single-marker bloodstream-form cell line was maintained under G418 selection (Wirtz et al., 1999 (link)). For trypanosome transfections 3×10⁷–4×10⁷ cells in mid-log phase (1×10⁶ cells/ml) were transfected with 10 µg of DNA using an AMAXA nucleofection system and the human T-cell nucleofection kit (Lonza). Stably transformed clonal cell lines were then selected by limiting dilution in the presence of appropriate antibiotics. Antibiotics used were G418 (2 µg/ml), hygromycin B (5 µg/ml), puromycin (0.2 µg/ml). COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and transiently transfected with Fugene HD reagent.

Manna P.T., Gadelha C., Puttick A.E, & Field M.C. (2015). ENTH and ANTH domain proteins participate in AP2-independent clathrin-mediated endocytosis. Journal of Cell Science, 128(11), 2130-2142.

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Silencing CEBPB in T Cells Modulates DC-Mediated Immune Responses

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DCs were enriched from PBMCs using the Pan-DC enrichment kit (Miltenyi, 130-100-777), and enriched DCs were stained with BV421 anti-human CD11c (BioLegend, 301628, 5 μl per sample), APC Cy7 anti-human HLA-DR (BioLegend, 307618, 5 μl per sample), FITC anti-human CD3 (BD Biosciences, 555232, 10 μl per sample) and PE anti-human CD123 (BD Biosciences, 340545, 5 μl per sample) antibodies. T cells were sorted from the non-DC fraction as CD3⁺ cells. DCs sorted as CD3⁻CD11c⁺HLA-DR⁺CD123⁻ were silenced and 48 h cells were stimulated with LPS (100 ng ml⁻¹) and 10 μg ml⁻¹ of OVA 323-339 (SP-51023-1, Genemed) in the presence of propyzamide (10 μM) or vehicle. After 6 h, cells were washed and co-cultured with allogenic T cells at a 1:10 ratio for 48 h. Cytokines were quantified in the supernatants using the LEGENDplex Th cytokine panel (BioLegend, 741027). CEBPB was silenced on T cells using the Human T cell nucleofection kit (Lonza, VPA-1002) and the Accell Human CEBPB-siRNA smart pool (Dharmacon, E-006423-00-0005). T cells were then activated by plate-bound anti-human CD3 (Thermo Fisher Scientific, 16-0037-85) and soluble anti-human CD28 (Thermo Fisher Scientific, 16-0289-85) in presence of propyzamide (10 μM) or vehicle.

Sanmarco L.M., Chao C.C., Wang Y.C., Kenison J.E., Li Z., Rone J.M., Rejano-Gordillo C.M., Polonio C.M., Gutierrez-Vazquez C., Piester G., Plasencia A., Li L., Giovannoni F., Lee H.G., Akl C.F., Wheeler M.A., Mascanfroni I., Jaronen M., Alsuwailm M., Hewson P., Yeste A., Andersen B.M., Franks D.G., Huang C.J., Ekwudo M., Tjon E.C., Rothhammer V., Takenaka M., de Lima K.A., Linnerbauer M., Guo L., Covacu R., Queva H., Fonseca-Castro P.H., Bladi M.A., Cox L.M., Hodgetts K.J., Hahn M.E., Mildner A., Korzenik J., Hauser R., Snapper S.B, & Quintana F.J. (2022). Identification of environmental factors that promote intestinal inflammation. Nature, 611(7937), 801-809.

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