Multiset software

A total of 100 µL of fresh samples were added into flow-type sample tube 1 and tube 2. Twenty µL mouse anti-rabbit CD4-FITC/CD8-PE/CD3-PCS mixed antibody (Beckman Coulter, USA) was added to tube 1. Twenty µL of the same type of control antibody was added to tube 2, incubated at 4°C for 30 min in the dark and then washed with phosphate buffered saline (PBS) buffer. The supernatant was removed and this step was repeated 2 times, centrifuged at 60 g for 10 min at 4°C. The red cell lysis solution was added to tubes 1 and 2 (each tube 300 µL). Light was avoided for 20 min at 4°C, washing was repeated 2 times, and PBS 200 µL cell liquid suspension was added. Tube 2 was used as a negative control to adjust the parameters, and tube 1 was used for detection. Lymphocyte gating was selected according to cell size. Next, using the CD3+ cells as gating, the percentage of CD4+, CD8+ T lymphocyte subsets could be obtained. The distribution of CD4+ CD25+ Foxp3+ lymphocyte subsets was detected using mouse anti rabbit CD4-FITC/CD25-PE antibody mix (Beckman Coulter), and the remainder of the procedure followed the above methods. The fluorescence parameters were obtained and analyzed with MultiSET software (Becton Dickinson Bio-sciences, USA).

Immunophenotyping was performed using a FACS Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, New Jersey, USA). Samples were analyzed within 24 h of specimen collection. In brief, 20 µl of CD3^FITC/CD8^PE/CD45^perCP/CD4^APC or CD3 ^FITC/CD16+CD56 ^PE/CD45^PerCP/CD19 ^APC MultiTest reagents (Becton-Dickinson, USA) was mixed with 50 µl of whole blood and incubated in the dark at room temperature for 15 min. Red blood cells were then lysed by adding 450 µl of fluorescence-activated cell sorter lysing solution (Becton Dickinson, USA).The tubes were then incubated at room temperature for another 15 min. MultiSET software (Becton-Dickinson, USA) was used to perform the analysis.