2.5.1. Lymphocyte Apoptosis

The Annexin V-FITC apoptosis detection kit (BioVision, Milpitas, CA, USA) was applied to detect the lymphocyte apoptosis. After being gently mixed, 200 μL of the whole blood sample was transferred to a conical polypropylene test tube, and 4 mL of lysing buffer (Thermo Fisher Scientific, Waltham, MA, USA) was added. The solution was incubated at room temperature for 7 min before being centrifuged at 1500× *g* for 5 min at 20 ◦C, and the supernatant was then discarded. Subsequently, 3 mL of phosphate buffered saline (PBS) (Corning®, Corning, NY, USA) was added to wash the cells, the solution was centrifuged for 5 min, and the supernatant was then discarded. Lymphocytes were suspended with 300 μL of binding buffer. Thereafter, 3 μL of Annexin V-FETC and propidium iodide were added, and the solution was incubated in the dark at room temperature for 5 min. Finally, samples were gently drawn and then the subject to analysis by the BD FACSCaliburTM flow cytometry (Becton Dickson, San Jose, CA, USA).
