Apc conjugated mouse igg κ isotype ctrl

For peptide cross-presentation experiments, 1.6 × 10⁶ Jaws II cells were incubated with 50 nmol of GRKKRRQRRRPQRWEKISIINFEKL, cholesterol-CSIINFEKL, or SIINFEKL peptide. After 4 hr of incubation, cells were washed and stained with either allophycocyanin (APC)-conjugated anti-mouse H-2K^b bound to SIINFEKL (141606, BioLegend) or APC-conjugated mouse IgG κ isotype Ctrl (400119, BioLegend), and the samples were analyzed by flow cytometry. For PeptiENV cross-presentation experiments, 15 nmol of either GRKKRRQRRRPQRWEKISIINFEKL or cholesterol-CSIINFEKL were complexed with 4.8 × 10⁷ PFU of VACV, and after the complexation, unbound peptides were removed by ultracentrifugation (20,000 × g, 50 min) through 36% sucrose cushion in 1 mM Tris (pH 9.0), and the purified viral pellets were suspended in 100 μL of complete RPMI-1640. 2 × 10⁶ BMDCs were plated in 2 mL of complete RPMI-1640 media and were infected with the purified PeptiENV viruses. After 4 hr of incubation, cells were washed and stained with either APC-conjugated anti-mouse H-2K^b bound to SIINFEKL (141606, BioLegend) or APC-conjugated mouse IgG κ isotype Ctrl (400119, BioLegend), and the samples were analyzed by flow cytometry.