Bv421 conjugated anti cd4

Intracellular cytokine staining: CD4+ T cells secreting IL-4 and IFN-γ were detected from single cell suspension of spleen in mice 4 d post the HRSV challenge. A total of 2×10 6 splenocytes were added to each well and stimulated with 10 µg/mL F protein expressed by 293F cells for 12 h. Then, 1 µg/mL of the protein transport inhibitor brefeldin A (BD Biosciences, USA) was added to each well 5 h before the end of stimulation. The splenocytes were pre-incubated with Fc blocker (BD Biosciences), a monoclonal antibody against CD16-CD32, on ice for 10 min, and washed with FACS buffer. The cells were then labeled with mouse PE-conjugated anti-CD3, BV421-conjugated anti-CD4, BV510conjugated anti-CD44 and FITC-conjugated anti-CD62L (BD Biosciences) surface markers for 30 min on ice in the dark. The cells were fixed with Cytofix/Cytoperm (BD Biosciences), then permeabilized by 1X permeabilization buffer (BD Biosciences). APC-conjugated anti-IFN-γ and PE-conjugated anti-IL-4 antibodies (BD Biosciences) were incubated with cells at 4 °C for 30 min. In order to determine the accuracy of positive CD4+ T cells, isotype IgG control antibodies were used for APC-conjugated anti-IFN-γ and PE-conjugated anti-IL-4. Moreover, lymphocytes stimulated by PMA and Ionomysin were used to verify the accuracy of flow cytometry and the rationality of flow gate.