Cytoperm fixation permeabilization solution

ICS assay was performed to detect antigen-specific CD4+ T cell and CD8+ T cell immune responses [14 (link)]. The splenocytes from each immunized C57BL/6 mice were cultured with RPMI 1640 containing 10% FBS in 96-well flat-bottom plates (2 × 10⁶ cells/well). Meanwhile, 0.1 MOI of VACV was diluted in RPMI 1640 containing 10% FBS. The cultured splenocytes were stimulated with 0.1 MOI of VACV (100 μl/well) for 6 h and then incubated with Golgiplug (BD Biosciences) for an additional 12 h at 37°C, 5% CO2 incubator. The cells were washed with PBS containing 1% BSA to stain with anti-CD3, anti-CD4, and anti-CD8 surface markers (Biolegend) for 30 min at 4°C in the dark. After that, the cells were fixed and permeabilized using a BD Cytoperm fixation/permeabilization solution (BD Biosciences) and then stained with a cocktail of antibodies: IL-2 (Biolegend), IL-4 (Biolegend), INF-γ (Biolegend), TNF-α(Biolegend). Data were acquired on a BD FACSAria III flow cytometer (BD Biosciences) and analyzed with FlowJo 10.6.2.

ICS assay was performed to detect antigen-specific CD4+ T cell and CD8+ T cell immune responses [14 (link)]. The splenocytes from each immunized C57BL/6 mice were cultured with RPMI 1640 containing 10% FBS in 96-well flat-bottom plates (2 × 10⁶ cells/well). Meanwhile, 0.1 MOI of VACV was diluted in RPMI 1640 containing 10% FBS. The cultured splenocytes were stimulated with 0.1 MOI of VACV (100 μl/well) for 6 h and then incubated with Golgiplug (BD Biosciences) for an additional 12 h at 37°C, 5% CO2 incubator. The cells were washed with PBS containing 1% BSA to stain with anti-CD3, anti-CD4, and anti-CD8 surface markers (Biolegend) for 30 min at 4°C in the dark. After that, the cells were fixed and permeabilized using a BD Cytoperm fixation/permeabilization solution (BD Biosciences) and then stained with a cocktail of antibodies: IL-2 (Biolegend), IL-4 (Biolegend), INF-γ (Biolegend), TNF-α(Biolegend). Data were acquired on a BD FACSAria III flow cytometer (BD Biosciences) and analyzed with FlowJo 10.6.2.

HEK293T cells were seeded in 12 well plates (3 × 10⁵ cells/well) for transfection. mRNA (1 μg) was transfected into HEK293T cells with TransITmRNA (Mirus Bio) according to the Manufacturer's instructions. After 24–48 h, the HEK293T cells were collected and resuspended for the next detection. To detect protein expression, the cells were fixed and permeabilized using a BD Cytoperm fixation/permeabilization solution (BD Biosciences) and then stained respectively with each antigen monoclonal antibody expression supernatant in wash buffer for 30 min on ice escape antigen M1. HEK293T cells that expressed M1 were stained with His antibody PE (Miltenyi Biotec). Whereafter, cells were incubated with Alexa Fluor® 488 Goat Anti-Mouse IgG (H + L) (YEASEN) for 30 min on ice in the dark. Data were acquired on a BD FACSCanto flow cytometer (BD Biosciences) and analyzed with FlowJo 10.6.2.