Fixation permeabilization concentrate buffer

To monitor CD4⁺ Th subsets, mice were infected i.p. with 3.0 × 10⁷ PFU of JEV, sacrificed at 5 dpi, and splenocytes were prepared. Splenocytes were then cultured in 96-well culture plates (10⁶ cells/well) with PMA/Ionomycin (Th1 and Th17) in the presence of monensin (2 μM) for 5 h at 37 °C. The stimulated cells were washed twice with PBS and surface stained with FITC-anti-CD4 for 30 min at 4 °C and then washed twice with PBS containing monensin. After fixation, the cells were washed twice with permeabilization buffer (eBioscience) and stained with PerCP-anti-IFN-γ and APC-anti-IL-17α in permeabilization buffer for 30 min at room temperature. Finally, the cells were washed twice with PBS and fixed using fixation buffer. To monitor Treg cells, splenocytes were stained by surface staining for FITC-anti-CD4 markers for 30 min on ice and then fixed with fixation/permeabilization concentrate buffer (eBioscience) for 6 h at 4 °C. After fixation, the cells were washed twice with permeabilization buffer (eBioscience) and stained with PE-anti-Foxp3 in permeabilization buffer for 30 min at room temperature. The sample analysis was performed using a FACSCalibur flow cytometer.

To monitor CD4⁺ Th subsets, mice infected with JEV (3.0 × 10⁷ PFU) were sacrificed at 3 and 5 dpi, and splenocytes were prepared. Splenocytes were cultured in 96-well culture plates (10⁶ cells/well) with PMA (50 ng/ml) plus ionomycin (750 ng/ml) in the presence of monensin (2 μM) for 5 h at 37 °C. The stimulated cells were washed twice with PBS and surface stained with FITC-anti-CD4 for 30 min at 4 °C and then washed twice with PBS containing monensin. After fixation, the cells were washed twice with permeabilization buffer (eBioscience) and stained with PerCP-anti-IFN-γ and APC-anti-IL-17α in permeabilization buffer for 30 min at room temperature. Finally, the cells were washed twice with PBS and fixed using fixation buffer. To monitor Treg cells, splenocytes were surface stained for FITC-anti-CD4 markers for 30 min on ice and then fixed with fixation/permeabilization concentrate buffer (eBioscience) for 6 h at 4 °C. After fixation, the cells were washed twice with permeabilization buffer and stained with PE-anti-Foxp3 in permeabilization buffer for 30 min at room temperature. The sample analysis was performed with FACS Calibur flow cytometer.