Nucleofector™ Kits for Human T Cells

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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

PMNCs were purified by density gradient centrifusion with Ficoll‐paque Plus (GE Healthcare, Waukesha, WI, http://www3.gehealthcare.com/en/Global_Gateway) or BD Vacutainer CPT (BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com) according to the manufacturer's instructions. Three micrograms of expression plasmid mixture was electroporated into 3–5 × 106 PMNCs with the Nucleofector 2b Device (Lonza, Basel, Switzerland, http://www.lonza.com/) with an Amaxa Human T‐cell Nucleofector kit according to the manufacturer's instructions. The program used was V‐24. The cell viability and transfection efficiency of PMNCs in our lab were 70%–80% and 40%–60%, respectively. The plasmid mixtures used in the experiments are shown in Supporting Information Table 1. The cells equivalent to 3 × 104–1 × 106 of input cells were then seeded onto six‐well plates covered with a MEF feeder layer. For the induction from αβT cells, the transfected cells were cultured in X‐vivo10 (Lonza) supplemented with 30 U/ml IL‐2 (PeproTech Inc., Rocky Hill, NJ, http://www.peprotech.com/) and 5 μl/well of Dynabeads Human T‐activator CD3/CD28. For non‐T‐cell induction, αMEM medium containing 10% FBS, 10 ng/ml IL‐3, 10 ng/ml IL‐6, 10 ng/ml G‐CSF, and 10 ng/ml GM‐CSF was used. Two days after the transfection, an equal volume of Primate ESC medium containing bFGF and 10 μM Y27632 was added into each well without aspiration of the previous medium. The culture medium was then replaced with Primate ESC medium containing bFGF and Y27632 4 days after the transfection. The colonies were counted 16–25 days after plating, and the colonies similar to human ESCs were selected for further cultivation and evaluation. Generation of iPSC from CD34+ rich population was performed as described previously with slight modification [17]. Briefly, purified CD34+ cells from PMNC was expanded for 6 days in StemSpan SFEM medium (StemCell Technologies Inc, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 10 ng/ml IL‐3, 100 ng/ml IL‐6, and 300 ng/ml of Flt3 ligand, stem cell factor (SCF), and TPO. Three micrograms of the plasmid mixture was then electroporated into 1 × 105 cells. The cells equivalent to 0.2–6 × 104 of input cells were then seeded onto six‐well plates coated with Retronectin (Takara Bio Inc., Otsu, Shiga, Japan, http://www.takara.co.jp) in the StemSpan medium. The culture medium was gradually changed to the Primate ESC medium as described above. The iPSC clones used in this study are summarized in Supporting Information Table 2. We calculated the iPSC induction efficiency based on the number of iPSC colonies per number of seeded cells which were estimated from the number of cells used for the electroporation.

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