TALEN-Mediated T Cell Activation Assay

Prior to gene editing, activation of T cells was assessed by staining for CD25. Downstream experiments were performed only when T cells were highly activated (>85% CD25-expression). For small scale transfer of TALEN mRNAs, 1 × 10⁶ cells were harvested by centrifugation (300xg, 5 min) and the supernatant removed. Cells were resuspended in 50 µl CliniMACS ^® Electroporation Buffer (Miltenyi Biotec). Just before electroporation, 7.5 µg of each left and right (or left/left, right/right) TALEN pairs were mixed with the resuspended T cells and electroporated using a CliniMACS ^® Prodigy with Electroporator unit (Miltenyi Biotec) with the previously described Setting 3 (Alzubi et al., 2021 (link)). Post electroporation, cells were recovered in 400 µl of pre-warmed X-VIVO™ 15 (Lonza) medium supplemented with 200 U/ml rhIL-2 (Immunotools) and seeded into two wells of U-shaped 96-well plates. Half of the cells were subjected to a transient low temperature shift to 32°C for 24 h before being shifted to 37°C. The other half was directly cultured at 37°C. Approximately half of the media was changed every 2 days and the cells split every 3–4 days.