Immune cell isolation and flow cytometry

Venous blood was collected aseptically from patients and healthy volunteers. Red blood cells in the human peripheral blood were lysed and washed twice with phosphate-buffered saline (PBS). In addition, single-cell suspensions of blood and liver of mice were obtained 12 h after ConA administration by using mechanical and enzymatic dispersion as described previously^{15 (link)}. In general, the peripheral blood and liver were harvested. Red blood cells in the peripheral blood were lysed. A single-cell suspension of the liver was mechanically disrupted and then enzymatically digested with 1 mg/mL collagenase IV. Then, 1 × 10⁶ freshly prepared cells were suspended in 100 μl of PBS and stained with different combinations of fluorochrome-coupled antibodies. Data were acquired by flow cytometry on a FACS Canton II (BD Biosciences) or NovoCyte (ACEA) and analyzed by FlowJo software.