Mouse t cell nucleofector kit

Stat3-specific siRNA or Allstar negative control siRNA (Qiagen) was administrated to Th17 cells by mouse T cell nucleofector kit from AMAXA. Silencing of STAT3 in Th17 cells was verified by western blot on individual aliquots at 3 days following transfection. The remaining Stat3-silenced Th17 cells were then re-stimulated with indicated cytokine(s).

1 × 10⁷ OT-I CTLs (5–8 days after activation) were nucleofected 24 h prior to imaging with 5 μg Lifeact-mApple and probe construct plasmids using the Mouse T Cell Nucleofector Kit (Lonza). EL4-blue target cells were pulsed with 1 μM SIINFEKL for 30 min at 37°C, washed in serum-free CO₂-independent medium (with L-glutamine) and plated onto 35-mm glass-bottom culture dishes (MatTek) coated with 0.5 μg/mL murine ICAM-1/Fc. ∼2 × 10⁶ nucleofected CTLs in CO₂-independent T cell medium were added dropwise, imaging was started within 5 min. Interactions were imaged with cells in an environmental chamber maintained at 37°C (Okolabs) on an Olympus IX81 microscope (Olympus) using the Andor Revolution spinning-disc microscope with Yokogawa CSU-X1 spinning disk, iXon Ultra 888 EMCCD camera, 2x camera adaptor (Andor Technology, Oxford instruments) and Olympus Universal Plan Super Apochromat silicone immersion objective. 12-18 z stacks (0.8-μm apart) were imaged every 12 s with fluorophores excited at 405, 488, and 561 nm in each z-plane. 4D datasets were rendered and analyzed with Imaris software (Bitplane).

Knowkdown using siRNA in CD4⁺ T cells were performed as described by Ciofani et al.^{27 (link)}. CD4⁺ T cells from wild-type mice were cultured for 16 h under Th0 conditions, and 300 pmol of control or c-Jun siRNA (Invitrogen) was introduced using the Mouse T cell Nucleofector Kit (Lonza) according the manufacture’s instruction. After recovery by incubation in fully supplemented culture media (Lonza), the cells were cultured for 3 days under Th17 conditions. Sequences for c-Jun siRNAs are as follows: #1, 5′-UACUGUAGCCGUAGGCACCGCUCUC-3′; #2, 5′-AUGACUUUCUGCUUAAGCUGUGCCA-3′; #3, 5′-AAACGUUUGCAACUGCUGCGUUAGC-3′.