Amaxa mouse dc nucleofector kit

DNA vaccine (for skin immunization): plasmid DNA encoding xbp1 and hsp70 fused to TRP2 was described previously57 58 (link) and was purified using EndoFree plasmid kits (Qiagen) following vendor’s instructions (Qiagen) (Valencia, California, USA). GG bullets were made from plasmid DNA (120 µg) and gold microcarriers (60 mg; 0.6 or 1 µm diameter; Bio-Rad) per the vendor’s instruction and stored at 4°C in the presence of desiccant pellets.
DC vaccine: DC were generated from BM of naïve B6 WT mice (male or female, 6–8 weeks) as reported previously.57 58 (link) On day 6 of culture, CD11c⁺ DC (2×10⁶) purified from non-adherent cells using anti-CD11c microbeads (Miltenyi Biotec) (CD11c⁺ DC purity >95%) were transfected with 7 µg endotoxin-free vaccine DNA using an Amaxa mouse DC Nucleofector kit (Lonza) following the vendor’s instruction and cultured in RPMI 1640 media supplemented with 10%FBS, 2 mmol/L glutamine and 1× antibiotic/antimycotic solution without maturation factor(s). After 2 days, DC were gently collected and resuspended in endotoxic-free 1× PBS (Sigma) immediately prior to injection.