Clinimacs electroporation buffer

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.

PBMCs-derived T cells or CD4/8 selected T cells were thawed and recovered for 24 h with TexMACS GMP medium supplemented with 12.5 ng/mL of recombinant hIL-7 and 12.5 ng/mL of recombinant hIL-15. On the same day or 24 h later, T cells were activated with MACS GMP T cell TransAct (Miltenyi Biotec), according to the manufacturer’s instructions, for 3 days prior to electroporation. For TALEN mRNA transfer, 1 × 10⁶ activated T cells were electroporated with 7.5 μg of each TALEN mRNA, unless stated otherwise, in a final volume of 50 μL CliniMACS electroporation buffer (Miltenyi Biotec) in the test cuvette adaptor using the electroporation settings listed in Table 1.