Brefeldin a monensin golgi plug

10 µg of SOMV and 1 µg 9RE7 are used to synthesize SOMV-9RE7 as described above. E7-specific CD8+ T cell activation follows previously established protocol [24 (link), 25 (link)]. SOMV-9RE7 is incubated with 1 × 10⁵ dendritic cell line in 96 well plate cultured with complete RPMI media at 37 °C, 5% CO2 overnight. After aspirating culture medium and washing with PBS, 5 × 10⁵ E7-specific CD8+ T cells were added to the dendritic cell line and blocked with Brefeldin A + Monensin Golgi Plug (Thermo Fisher Scientific) overnight. Cells were collected and stained with APC-A750-conjugated anti-mouse CD8α antibody (Biolegend) before permeabilization with eBioscience Fixation (Invitrogen) and intracellular staining FITC-conjugated IFNγ antibodies.

To stain for intracellular cytokines, samples were incubated with PMA/Ionomycin cell stimulation cocktail and Brefeldin A + Monensin Golgi Plug from Thermo Fisher Scientific for 4 h at 37 °C. Cells were then collected and prepared as described in the flow cytometry section. Prior to intracellular staining, cells were permeabilized using eBioscience Foxp3 / Transcription Factor Staining Buffer Set from Thermo Fisher Scientific and stained for intracellular cytokines.