For each sample, 3 tubes containing the following combinations of monoclonal antibodies purchased from Southern Biotechnology Associates (Birmingham, AL, USA) were set up: 10 μl of CD4-phycoerythrin (106 cells/μg) and 10 μl of CD3-fluorescein isothiocyanate (106 cells/μg); 10 μl of CD8a-phycoerythrin (106 cells/μg) and 10 μl of CD3-fluorescein isothiocyanate (106 cells/μg); and 10 μl of IgG1-fluorescein isothiocyanate (106 cells/μl) and 10 μl of IgG1- phycoerythrin (106 cells/μl). Each tube was added 50 μl mononuclear cell suspension, gently mixed and then incubated at 4°C for 20 min in the dark. Then 500 μl of CMF-PBS solution was dispensed into each tube, gently mixed, and left for 10 min at room temperature out of direct light.