Amaxa mouse t cell nucleofector kit

Synthetic RNAs that correspond to canonical or di-uridylated miRNAs (Integrated DNA Technologies) were nucleofected into freshly isolated naïve CD4 T cells using the Amaxa Mouse T-cell Nucleofector Kit and Amaxa Nucleofector II (Lonza) according to the manufacturer's instructions.

Total T cells isolated from C57BL/6 wild-type mice by magnetic bead negative selection (Miltenyi Biotec) were transfected with 600 pmol siRNA using the Amaxa Mouse T cell Nucleofector kit and according to the manufacturer’s instructions (Lonza). Transfected cells were rested for 2 h before stimulation and assessment of proliferation as described previously. Chemically modified siRNAs were generated as previously described (Mantei et al., 2008 (link)) and their sequences are as follows: 5′-3′ nontargeting control, sense 5′-GmGmAGCGCACCAUCUUCTdCdAdAmUmUm-3′, antisense 5′-AdUUGAGAAGAUGGUGCGCUCmCm-3′; Usp9X (1) 5′-AmAmCCAAGUAACUCAUGATdCdAdAmGmCm-3′, antisense 5′-TdUGAUCAUGAGUUACUUGGUmUm-3′; Usp9X (2), sense 5′-CmCmCAAAUGAAGAAGUGACdAdAdAmAmAm-3′, antisense 5′-TdUUGUCACUUCUUCAUUUGGmGm-3′, Usp9X (3), 5′-UmUmUGAAUUUCCUCGAGAGdTdTdAmGmAm-3′, antisense 5′-TdAACUCUCGAGGAAAUUCAAmAm-3′; Usp9X (4), 5′-CmUmUGGCAAAGUUAGAUGAdTdAdUmGmAm-3′, antisense 5′-AdUAUCAUCUAACUUUGCCAAmGm-3′. Cm, Um, Am, Tm, or Gm denote a methoxy nucleotide and Gd, Ad, Td, or Cd denote a deoxynucleotide.

Freshly purified spleen CD8+ T cells were transfected with either
3.6 μg pGL3 basic vector or 3.6 mg Wnt10b-luc
(including WT or mutant DNA segments) reporter constructs together with 0.4
μg TK-pRL transfection control vector using the Amaxa Nucleofector
system and Amaxa Mouse T Cell Nucleofector Kit (Lonza). Cells were left
unstimulating for 24 h and cultured with ionomycin (0.5 mg/mL, Sigma) plus
TGFβ1 (5 ng/mL, Biolegend) for another 24 h. Luciferase activity was
determined by the Dual-Luciferase reporter assay kit (Promega BioSciences,
San Luis Obispo, CA).