Amaxa nucleofector solution

Untouched CD4 T cells were isolated from PBMCs of HDs through immunomagnetic separation with CD4⁺T‐cell isolation kit (Miltenyi Biotec) according to manufacturer's instructions. Cells were resuspended at room temperature in Amaxa Nucleofector solution (Lonza, Basel, Switzerland), mixed with a solution of 500 pmol FAM‐labelled oligomers, transferred into cuvettes and nucleofected by performing V‐024 program in Amaxa Nucleofector 2b device (Lonza). The following oligomers were used, based on previous literature¹¹: ISG15‐siRNA #9 (5′‐GGA CAA AUG CGA CGA ACC U‐3′) and #12 (5′‐GCA ACG AAU UCC AGG UGU C‐3′), and a scrambled siRNA as non‐targeting control (5′‐GCC GAU CGU CGA GAC UAA U‐3′). After nucleofection, cells were collected in prewarmed complete medium. Dead cells were removed from samples with Dead cell removal kit (Miltenyi Biotec) and the recovered cells were cultured with Dynabeads (Gibco Thermo Fisher Scientific) at a 1:2 bead‐to‐cell ratio, in presence or absence of 500 IU mL⁻¹ recombinant IFNα for 18 h.

A transient AKT siRNA knockdown was performed in Raji and Raji 4RH cells. Cells were cultured in RPMI1640 with 10% HIFBS at 37° C in 5% CO₂. Cells were passaged 3 days before experimental start and in log rhythmic growth phase. 2 × 10⁶ cells were pelleted, washed and combined with 100uL Amaxa Nucleofector Solution (Lonza, Walkersville MD, USA) and then transferred to an Amaxa cuvette where siRNA was added at a concentration of 300 nM/sample. Samples were then electroporated using Amaxa Nucleofector Devise (Lonza, Walkersville MD, USA) using the M-13 protocol. After electroporation cells were transferred back into media and allowed to recover and presence of siRNA knockdown was detected via western blot.