Magnetic t cell negative selection kit

For T cell stimulation assays, spleen or dLN were harvested from uninfected C57BL/6J mice. Pan-T cells were enriched with magnetic T cell negative selection kit (Stem Cell Technologies, Vancouver, Canada) from healthy mouse splenocytes. Single cell suspensions from spleen were either lysed for red blood cells and plated whole or enriched for T cells as described above; dLN cells were plated whole or enriched for T cells as described above. Bulk cells or purified T cells were then cultured at 1e6 cells/well in 200ul in 96-well plates. Both groups were cultured for 24 hours in the presence of recombinant IFNα (Pacific BioLabs; 50U/ml), recombinant IFNβ (Pacific BioLabs; 50U/ml), and recombinant IL-2 (Biolegend; 20ng/ml). Golgiplug (BD Biosciences) was added at 1:1000 after 20 hours of culture. For TCR stimulation positive control wells, 96-well plates were coated with 1ug/ml purified anti-CD3 and 2ug/ml anti-CD28 (Biolegend) for 24hours at 4 degrees C before plating cells. Negative controls were cultured in media alone.