Human t cell nucleofector solution

Manufactured by Lonza

Sourced in Germany

The Human T Cell Nucleofector Solution is a specialized electroporation reagent designed for the efficient transfection of human T cells. It is intended for use with the Nucleofector Device to facilitate the introduction of nucleic acids, such as plasmids or small interfering RNAs, into primary human T cells.

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Lab products found in correlation

Hygromycin b, by Thermo Fisher Scientific (3 mentions) Cd3 cd28 dynabead, by Thermo Fisher Scientific (2 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions) Amaxa nucleofector 2, by Lonza (1 mentions) Puromycin, by Merck Group (1 mentions) Geneticin, by Merck Group (1 mentions) Amaxa technology, by Lonza (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) U 015 program, by Lonza (1 mentions) Cell line nucleofector solutions 5, by Lonza (1 mentions) Blasticidin, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Nucleofector solution (55 citations)

8 protocols using human t cell nucleofector solution

Genetic Manipulation of Leishmania major

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For the LmjF.22.0600 overexpression, 3 × 10⁷L. major cells in logarithmic phase were transfected with 10 µg of the recombinant vector pTH6nGFPc containing the LmjF.22.0600 sequence, in 100 µl of Human T Cell Nucleofector solution (Lonza, Basel, Switzerland).
For the LmjF.22.0600 deletion, 10⁷L. major cells expressing Cas9 and T7 (kind gift from Eva Gluenz, University of Oxford) in logarithmic phase were transfected with 30 µg of purified PCR. The transfection buffer was composed of 90 mM sodium phosphate, 5 mM potassium chloride, 0.15 mM calcium chloride, 50 mM HEPES, pH 7.3. The same transfection without purified PCR was used as control.
The transfections were performed in 2 mm gap cuvettes (Lonza) with program X-001 of the Amaxa Nucleofector II (Lonza). Transfected cells were immediately transferred into 5 ml pre-warmed medium and left to recover overnight at 26 °C before adding 30 µg/ml hygromycin B (Thermo Fisher Scientific) for the overexpression and two drugs for the deletion: geneticin (Sigma-Aldrich) at 20 µg/ml and puromycin (Sigma-Aldrich) at 30 µg/ml.

Basmaciyan L., Azas N, & Casanova M. (2019). A potential acetyltransferase involved in Leishmania major metacaspase-dependent cell death. Parasites & Vectors, 12, 266.

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Silencing LAT1 in Human T Cells

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The siRNA-mediated downregulation of LAT1 expression in human T cells was performed using the Amaxa technology (Lonza, Cologne, Germany). A total of 5 × 10⁶ primary, unstimulated human T cells were washed twice with 1× PBS. T cells were then resuspended in 100 µL human T cell Nucleofector solution (Lonza, Cologne, Germany) before electroporation with 2 µg hLAT1 siRNA (sense: CUCUUUGCCUAUGGAGGAU[dT][dT], Sigma Aldrich) using the Nucleofector™ 2b Device, program V 24 (Lonza). As controls, T cells were either not electroporated or electroporated in the presence of 2 µg non-target RNA (negative control CSR CL 000-005, Eurogentec, S.A.), or without any siRNA. After electroporation, cells were incubated for 5 h in 3 mL prewarmed AIM V^® culture medium (Life Technologies, Carlsbad, CA, USA) to recover before further treatment.

Werner A., Koschke M., Leuchtner N., Luckner-Minden C., Habermeier A., Rupp J., Heinrich C., Conradi R., Closs E.I, & Munder M. (2017). Reconstitution of T Cell Proliferation under Arginine Limitation: Activated Human T Cells Take Up Citrulline via L-Type Amino Acid Transporter 1 and Use It to Regenerate Arginine after Induction of Argininosuccinate Synthase Expression. Frontiers in Immunology, 8, 864.

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Genetic Manipulation of Y-strain Epimastigotes

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Y-strain epimastigotes were grown for 4 days under standard conditions, collected at 1600 ×g for 10 min, washed once with buffer-A with glucose (BAG), and resuspended in 100 μl of Human T Cell Nucleofector Solution (Lonza). 5 × 10⁷ cells were mixed with 5 μg of each sgRNA plasmid with or without 5 μg of DNA donor and electroporated under the U-033 Amaxa Nucleofector protocol. Cells were allowed to recover overnight and appropriate antibiotics for selection (250 μg/ml G-418 and 15 μg/ml blasticidin) were added.

Dave N., Cetiner U., Arroyo D., Fonbuena J., Tiwari M., Barrera P., Lander N., Anishkin A., Sukharev S, & Jimenez V. (2021). A novel mechanosensitive channel controls osmoregulation, differentiation, and infectivity in Trypanosoma cruzi. eLife, 10, e67449.

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Electroporation of Leishmania promastigotes

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Logarithmic L. major promastigotes were harvested by centrifugation at 600xg for 10min, washed once in sterile PBS and resuspended at 3x10⁷cells/mL in 100μL of Human T Cell Nucleofector solution (Lonza, Basel, Switzerland). Cells were transferred to Amaxa electroporation cuvettes maintained at 4°C containing 10μg of DNA. Cells were then electroporated with the program U-033 on the Nucleofector machine (Amaxa GmbH, Cologne, Germany). Following electroporation, cells were incubated overnight in their culture medium and transfectants were selected with 30μg/mL hygromycin B (Life Technologies).

Basmaciyan L., Robinson D.R., Azas N, & Casanova M. (2019). (De)glutamylation and cell death in Leishmania parasites. PLoS Neglected Tropical Diseases, 13(4), e0007264.

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Optimized SB CAR T Cell Production

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SB CAR T cells were produced by adapting the previously reported method to conventional T cells.⁶¹ (link) Human T cells were purified with EasySepTM beads and resuspended in Human T cell Nucleofector Solution (Lonza). For each cuvette, 5 × 10⁶ purified T cells were electroporated (program U-14) in presence of the pT4.iC9.79D vector and the transposase SB100X plasmid (Addgene 34879) or of the transposase SB100X mRNA. After electroporation, T cells were activated with CD3/CD28 Dynabeads (Thermo Fisher Scientific) and stimulated with 80 U/mL IL-2 (Peprotech, Rocky Hill, NJ, USA). CAR T cells were cultured for 10 days before cryopreservation or immediate use.

Magnani C.F., Myburgh R., Brunn S., Chambovey M., Ponzo M., Volta L., Manfredi F., Pellegrino C., Pascolo S., Miskey C., Ivics Z., Shizuru J.A., Neri D, & Manz M.G. (2023). Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells. Molecular Therapy Oncolytics, 30, 56-71.

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Optimized Transfection Protocols for Various Cell Types

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3.10⁶ 293T cells per condition were transfected with 10 μg of plasmid DNA into 10 cm Petri dishes using lipofectamine 2000 (Invitrogen) according to the protocol provided by the supplier. The cells were used 48 h later for further experiments.
5.10⁵ HBMEC per cuvette were washed in PBS and resuspended in 100 μl of Cell Line Nucleofector Solutions V (Lonza). These cells were transfected by nucleofection using the U-015 program (Lonza) with 3–4 μg of DNA.
2.10⁶ or 5.10⁶ CEM cells per condition were washed in PBS and resuspended in 100 μl of Cell Line Nucleofector Solutions V (Lonza). These cells were transfected by nucleofection using the C-016 program (Lonza) with 5 or 10 μg of DNA, respectively.
10.10⁶ human primary T cells per condition were washed in PBS and resuspended in 100 μl of Human T Cell Nucleofector Solution (Lonza). These cells were transfected by nucleofection using the U-014 program (Lonza) with 10 μg of DNA.
All cells transfected by nucleofection were used for functional assays after an overnight incubation time.

Megrelis L., El Ghoul E., Moalli F., Versapuech M., Cassim S., Ruef N., Stein J.V., Mangeney M, & Delon J. (2018). Fam65b Phosphorylation Relieves Tonic RhoA Inhibition During T Cell Migration. Frontiers in Immunology, 9, 2001.

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Engineered anti-CD117 CAR T cells

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CAR T cells were produced by electroporation of purified T cells from healthy donors (Blutspende Zurich, Switzerland). T cell activation was performed with CD3/CD28 Dynabeads (Thermo Fisher Scientific) and 80 U/mL IL-2 (Peprotech, Rocky Hill, NJ, USA). mRNA anti-CD117 CARs were electroporated in T cells after 4 days post activation. Briefly, 5 × 10⁶ T cells were resuspended in Human T cell Nucleofector Solution (Lonza) and electroporated in the presence of the anti-CD117 CAR mRNA using the Amaxa program T-20.

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Genetic Modification of Leishmania major

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Logarithmic L. major promastigotes were harvested by centrifugation at 600 × g for 10 min, washed once in sterile PBS and resuspended at 3 × 10⁷ cells/ml in 100 μl of Human T Cell Nucleofector solution (Lonza, Basel, Switzerland). Cells were transferred to Amaxa electroporation cuvettes maintained at 4 °C and already containing 10 μg of DNA. Cells were then electroporated with the program U-033 on the Nucleofector machine (Amaxa GmbH, Cologne, Germany). Following electroporation, cells were incubated overnight in their culture medium and transfectants were selected with 30 μg/ml hygromycin B (Life Technologies, France) for single transfection and with 30 μg/ml hygromycin B and 15 μg/ml blasticidin (Life Technologies, France) for double transfections.

Casanova M., Gonzalez I.J., Sprissler C., Zalila H., Dacher M., Basmaciyan L., Späth G.F., Azas N, & Fasel N. (2015). Implication of different domains of the Leishmania major metacaspase in cell death and autophagy. Cell Death & Disease, 6(10), e1933-.

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