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T cell nucleofector kit

Manufactured by Lonza
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The T cell nucleofector kit is a laboratory product designed for the electroporation of primary human T cells. It enables the efficient delivery of nucleic acids, such as plasmids or small interfering RNAs, into T cells for various research applications.

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Lab products found in correlation

Amaxa electroporator, by Lonza (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Lymphocyte separation medium, by MP Biomedicals (1 mentions) 4d nucleofector system, by Lonza (1 mentions) Nucleofector 2 device, by Lonza (1 mentions)

Close spelling variants of this product

Nucleofector Kits for Mouse T Cells Nucleofector Kits for Human T Cells Nucleofector Kit T Nucleofector kit

6 protocols using t cell nucleofector kit

1

CD4+ T Cell Isolation and Knockdown

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Primary PBMCs were isolated from human blood by density centrifugation using a lymphocyte separation medium (MP Biomedicals, Santa Ana, CA, USA). CD4+ T cells were negatively selected from PBMCs using the CD4+ T cell isolation kit (Miltenyi Biotec, Gaithersburg, MD, USA). Cell purity of more than 95% was obtained after CD4+ T cell isolation. For knockdown experiments, Accell control siRNA and SMARTpool Accell siRNAs targeted IGFBP1 or PYY (Dharmacon, Lafayette, CO, USA) and were transfected with Accell siRNA delivery media or T cell nucleofector kit according to manufacturer’s instructions (Lonza, Bend, OR, USA). To increase cell viability, CD4+ T cells were transfected with T cell nucleofector kit (Lonza) and maintained in media supplemented with 50 ng/mL IL-2 (Peprotech, Cranbury, NJ, USA) for the first 24 h. The IL-2 media was then replaced with regular RPMI media before the T cell receptor-mediated activation. THP1 human monocytes and H9 human T lymphocytes (derivative of HuT78) obtained from ATCC were maitained in RPMI 1640 media supplemented with 25 mM HEPES, 10% heat-inactivated FBS and penicillin/streptomycin.
Han K., Singh K., Rodman M.J., Hassanzadeh S., Baumer Y., Huffstutler R.D., Chen J., Candia J., Cheung F., Stagliano K.E., Pirooznia M., Powell-Wiley T.M, & Sack M.N. (2021). Identification and Validation of Nutrient State-Dependent Serum Protein Mediators of Human CD4+ T Cell Responsiveness. Nutrients, 13(5), 1492.
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2

Stable Transfection of PF Cell Lines

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Cell lines were generated as previously reported [82 (link),83 (link)]. Briefly, 107 PF cells (strain 29–13) were transfected with 10 μg MfeI-linearised pC-F1α-GFP-PTP vector or BsmI-linearised pC-F1β-GFP-PTP vector using an Amaxa electroporator and T-cell nucleofector kit (Lonza) as per the manufacturer protocol. Stable transformants were selected in and maintained in full growth medium supplemented with 2 μg/mL puromycin at 27°C with 5% CO2.
Tulloch L.B., Menzies S.K., Fraser A.L., Gould E.R., King E.F., Zacharova M.K., Florence G.J, & Smith T.K. (2017). Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues. PLoS Neglected Tropical Diseases, 11(9), e0005886.
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3

Cytotoxic T Cell Nucleofection with RUSH-GZMB

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At day 6, count the CTLs obtained and determine cell density. CTL nucleofection with the RUSH-GZMB construct is performed using the Amaxa T cell nucleofector kit, with the following protocol:
Capitani N., Cassioli C., Ravichandran K, & Baldari C.T. (2023). Exploiting the RUSH System to Study Lytic Granule Biogenesis in Cytotoxic T Lymphocytes. Methods in molecular biology (Clifton, N.J.), 2654, 421-436.
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4

CXCR4 Gene Editing in Primary T Cells

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The stimulated primary human or Rhesus macaque CD4+ T cells were electro-transfected with CXCR4 gRNAs-Cas9 or control plasmids using the T cell Nucleofector kit #VPA-1002 (Amaxa Human T cell Nucleofector kit) or P3 Primary Cell 4D-Nucleofector × Kit (V4XP-3012). In brief, 5 × 106 CD4+ T cells per sample were collected and washed twice in PBS. The cells were re-suspended in 100 μl Nucleofector Solution with 2 μg of plasmids respectively. The cell/DNA mixture was transferred into the certified cuvette and electro-transfected with an Amaxa Nucleofector II Device using the Nucleofector Program T-020 or a Lonza 4D-Nucleofector System. After transfection, the cells were cultured in RPMI 1640 medium supplemented with 10% FBS for two days and then used for further analysis. Cells were counted using trypan blue dye exclusion method on a hemocytometer under a microscope.
Hou P., Chen S., Wang S., Yu X., Chen Y., Jiang M., Zhuang K., Ho W., Hou W., Huang J, & Guo D. (2015). Genome editing of CXCR4 by CRISPR/cas9 confers cells resistant to HIV-1 infection. Scientific Reports, 5, 15577.
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5

Cytotoxic T Cell Nucleofection with RUSH-GZMB

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At day 6, count the CTLs obtained and determine cell density. CTL nucleofection with the RUSH-GZMB construct is performed using the Amaxa T cell nucleofector kit, with the following protocol:
Capitani N., Cassioli C., Ravichandran K, & Baldari C.T. (2023). Exploiting the RUSH System to Study Lytic Granule Biogenesis in Cytotoxic T Lymphocytes. Methods in molecular biology (Clifton, N.J.), 2654, 421-436.
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6

Efficient Generation of iPSC Lines

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Patient enrollment, clinical data collection, blood sampling, and reprogramming and QC of iPSC lines has been described previously.5 (link),7 (link),9 (link) Briefly, 5×106 PBMCs isolated from the buffy coat of patient blood samples were nucleofected with plasmids pEP4 E02S ET2K,46 (link) pCXLE-hOCT3/4-shp53-F,47 (link) pCXLE-hUL,47 (link) pCXLE-hSK,47 (link) and pCXWB-EBNA148 (link) using the Amaxa Human T cell Nucleofector Kit and Nucleofector 2D Device. Following nucleofection, cells were resuspended in either T cell media (X-VIVO 10 media supplemented with 30U/mL IL-2 and 5 μl/well Dynabeads Human T-activator CD3/CD28) or non-T cell medium (αMEM supplemented with 10%heat inactivated FBS, 10 ng/ml IL-3, 10 ng/ml IL-6, 10 ng/ml G-CSF, and 10 ng/ml GM-CSF), plated onto mitomycin treated mouse embryonic fibroblasts, and placed in a 37°C incubator. Two days after nucleofection, 2 mL/well of Primate ES Cell Medium supplemented with 5 ng/ml bFGF was added to the wells. Stem cell-like colonies, typically appearing between days 25–32, were mechanically isolated and plated onto Matrigel-coated plates in mTeSR1. Quality control of iPSC lines was carried out as reported5 (link),7 (link) and consisted of karyotyping, pluripotency testing, EBNA-related gene analysis, and short tandem repeat (STR) confirmation of cell identity.
Workman M.J., Lim R.G., Wu J., Frank A., Ornelas L., Panther L., Galvez E., Perez D., Meepe I., Lei S., Valencia V., Gomez E., Liu C., Moran R., Pinedo L., Tsitkov S., Ho R., Kaye J.A., Thompson T., Rothstein J.D., Finkbeiner S., Fraenkel E., Sareen D., Thompson L.M, & Svendsen C.N. (2023). Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects. Neuron, 111(8), 1191-1204.e5.
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