Facs symphony a3 flow cytometer

The lung and spleen T-cell isolation and activation in immunized mice before and after infections were described as previously^{69 (link)}. To determine in vitro activation, single cells (2 × 10⁶) from the lung or spleen of euthanized mice were seeded in 24 well-cell culture plates and stimulated with endotoxin-free PspA protein (20 μg/mL) for 48 hours. Then, a 1 × brefeldin-A and monensin cocktail (1:1 ratio) was added to block Golgi-mediated cytokine secretion for 2–3 hours before cell collection. To determine in vivo activation, single cells (2 × 10⁶) isolated from the lung and spleen of euthanized mice on 2 DPSI were induced with 50 ng/mL phorbol myristate acetate and 1 μg/mL ionomycin for 1 h and then treated with the 1 × brefeldin-A and monensin cocktail for another 2 hours. Induced cells were harvested and resuspended in flow cytometry staining (FACS) buffer containing Fc block (anti-mouse CD16/32 antibodies) (1:200) for 10 min on ice. T cells were stained with fluorochrome-labeled anti-CD3, CD4, CD8, IFN-γ, TNF-α, and IL-17A mAbs (SI Table 3) and were analyzed by BD FACS symphony A3 flow cytometer (BD Biosciences) using FACSDiva software. Also, we included fluorescence minus one control staining to help identify gating boundaries. Flow data were analyzed using FlowJo v10.7.