Percp cyanine5.5 anti cd3

In order to detect the expression of IFN-γ and IL-4 in transfected naive CD4⁺ T cells after immunization, the cells were surface stained with PerCP-Cyanine5.5-anti-CD3 (Thermo Fisher Scientific Co., Ltd, Xian, China) and APC-anti-CD4 antibodies (Thermo Fisher Scientific Co., Ltd, Xian, China). Next, the cells were treated with fixation buffer (BioLegend, Inc, California, USA.) and permeabilization buffer (BioLegend, Inc, California, USA.) followed by intracellular staining with fluorochrome-conjugated PE-anti-IFN-γ (Thermo Fisher Scientific Co., Ltd, Xian, China) and PE-anti-IL-4 (Thermo Fisher Scientific Co., Ltd, Xian, China). When assaying CD4⁺ T cells from immunized mice, staining is performed using PerCP-Cyanine5.5-anti-CD3 (Thermo Fisher Scientific Co., Ltd, Xian, China), APC-anti-CD4 antibodies (Thermo Fisher Scientific Co., Ltd, Xian, China), PE-anti-IFN-γ (Thermo Fisher Scientific Co., Ltd, Xian, China), and BV605-anti-IL-4 (Thermo Fisher Scientific Co., Ltd, Xian, China). The degree of cell activation after transfection has been determined by staining the surface of naive CD4⁺ T cells with PE-anti-CD25 and FITC-anti-CD69 antibodies (Thermo Fisher Scientific Co., Ltd, Xian, China). The stained cells were measured by a BD FACSCelesta instrument (Beijing, China) and the results were analyzed by FlowJo.