Flow Cytometry Analysis of T Cell Subsets and Cytokines

The percentages of CD4⁺ and CD8⁺ T lymphocytes subsets and the production of cytokine in the splenocytes of immunized mice were determined using flow cytometry techniques as described previously (Zhang et al., 2015b (link); Wang S. et al., 2017 (link)). Briefly, splenocytes (2 × 10⁶cells/ml) of vaccinated mice were stimulated with G10E peptides (10 μg/mL) for 72 h. Then the suspensions were stained with anti-mouse CD3-APC, anti- mouse CD4-FITC and anti-mouse CD8-PE (eBiosciences, USA) for 30 min at 4 °C in the dark.
Splenocytes (2 × 10⁶ cells/ml) were also stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail (eBiosciences, USA) containing Phorbol myristate acetate (PMA, 20 ng/ml), Ionomycin (2 μg/ml), Brefeldin A (1 μg/ml), and Monensin (1 μg/ml) to inhibit the secretion of cytokine into the extracellular space. The cells were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit in accordance with the manufacturer's protocol (eBiosciences, USA) and then stained directly with anti-mouse CD4-FITC, anti-mouse CD8-PE, anti-mouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), anti-mouse IL-4 (APC), and anti-mouse IL-10 (PerCP-Cyanine5.5) (eBiosciences, USA) for 30 min at 4°C. All these cell population were analyzed by Cytoflex S Flow Cytometer (Beckman Coulter, USA) and the data were analyzed by CytExpert software.