Nucleofector™ Kits for Human T Cells

Protocol tips

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	Entire supplement should be added to the Nucleofector™ Solution prior to use.

Protocol tips
Entire supplement should be added to the Nucleofector™ Solution prior to use.

Publication protocol

Cas9 RNP mediated editing and lentiviral transduction of primary human T cells
Cas9 RNP mediated editing of T cells was carried out as described3 with modifications. Primary human CD4+ or CD8+ T cells were thawed, rested for 24 hours in TCM followed by stimulation with plate-bound anti-CD3 (10 µg/mL; Tonbo Biosciences, clone: UCHT1) and soluble anti-CD28 (5 µg/mL; Tonbo Biosciences, clone: CD28.2) for 48 hours in T cell medium supplemented with IL-2 (30 U/mL). Electroporation was performed using the Amaxa P3 Primary Cell kit and 4D-Nucleofecter (Lonza). The recombinant S. pyogenes Cas9 used in this study expresses a C-terminal HA tag and two nuclear localization signal (NLS) peptides that facilitate transport across the nuclear membrane. The protein was expressed and purified as described37 and obtained from Macrolab, University of California, Berkeley. Cas9 was stored in 20 mM HEPES at pH 7.5, 150 mM KCl, 10% (v/v) glycerol, 1 mM TCEP at −80 °C.

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Manufacturer protocol

Download the product protocol from Lonza for Nucleofector™ Kits for Human T Cells below.

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