Detection of intracellular cytokine production of splenocyte in mice by flowcytometry after immunization. Spleen cells of mice (three mice each group) immunized with the SjGP-3 formulated by various adjuvants were harvested after three times immunization (on week 9). The isolated splenocytes from individual mice were first stimulated with PMA, ionomycin plus BFA. Splenocytes were stained extracellularly by PE-Cy5 labeled anti-CD3 and FITC labeled anti-CD8. After fixation and permeabilization, the spleen cells were divided into three tubes and stained intracellularly with PE conjugated Rat IgG1, anti-mouse IFN-γ antibody or anti-mouse IL-4 antibody, respectively. A FACScan flow cytometer with Cell Quest software was used for data acquisition and analysis. The percentage of PE positive cells in Rat IgG1 isotype control was less than 0.5% (data not shown).