To quantify leukocyte populations, erythrocytes were first lysed by resuspending 100 µl of whole blood in ten volumes of Red Blood Cell (RBC) Lysis Buffer (Thermo Fisher) at room temperature for 5 min. Cells were then washed twice with phosphate-buffered saline (PBS), and incubated in the Multitest 6-Color TBNK Reagent or Multitest CD3/CD8/CD45/CD4 mix (both from BD Biosciences) following the manufacturer’s manuals. After two PBS washes, cells were resuspended in 500 µl of ice-cold PBS. BD Trucount™ Absolute Counting Tubes, which carry known fluorescent beads, were applied as a standard to quantify the absolute cell number.
To evaluate regulatory T cells, blood leukocytes were stained with FITC-conjugated CD4 antibody, Violet 450-conjugated CD127 antibody, PerCP-Cy5.5-conjugated CD45 antibody, and APC-conjugated CD25 antibody (5 µg/ml each, BD Biosciences) for 15 min on ice in the dark. After two PBS washes, cells were resuspended in 500 µl of ice-cold PBS and then loaded on a BD FACSCanto Plus. Single cells were first gated between FSC-A and FSC-H. Intact cells were then gated among single cells based on FSC-A and SSC-A. Leukocyte populations were distinguished according to the surface marker staining. The data were assessed using the BD FACSDivaTM software. Isotype controls were also set to ensure specific staining of antibodies (Supplemental Fig. 1 ).
To evaluate regulatory T cells, blood leukocytes were stained with FITC-conjugated CD4 antibody, Violet 450-conjugated CD127 antibody, PerCP-Cy5.5-conjugated CD45 antibody, and APC-conjugated CD25 antibody (5 µg/ml each, BD Biosciences) for 15 min on ice in the dark. After two PBS washes, cells were resuspended in 500 µl of ice-cold PBS and then loaded on a BD FACSCanto Plus. Single cells were first gated between FSC-A and FSC-H. Intact cells were then gated among single cells based on FSC-A and SSC-A. Leukocyte populations were distinguished according to the surface marker staining. The data were assessed using the BD FACSDivaTM software. Isotype controls were also set to ensure specific staining of antibodies (