Facsc flow cytometer

PBMCs (5×10⁵/ml) were incubated for 20 minutes at 4? in the dark with human monoclonal antibodies specific for CD3, CD4, CD45RA, CD25, CD45RO, CD28, and CD62L. Antibodies labeled with fluorescence were obtained from BD Bioscience (San Diego, CA, USA), including phycoerythrin (PE)-conjugated antibodies for CD25 (#555432), CD45RA (#556627), CD45RO (#555493), and CD28 (#555729); Antibodies were obtained from Biolegend (San Diego, CA, USA), including fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (#344804), Percep/Cyanine5.5-conjugated anti-CD4 (#344608), and allophycocyanin (APC)-conjugated anti-CD62L (#304810), respectively. According to manufacturer's instruction, antibodies were diluted as 1:100 per sample and analysis were performed in triplicate. After incubation, cells were washed twice with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde. All analyses were performed using a BD FACSC flow cytometer with CellQuest software (BD PharMingen). Results were expressed as the percentages of gated lymphocytes. CD3⁺CD4⁺CD28^null T cells were characterized as total effector T cells, CD3⁺CD4⁺CD25⁺CD62L⁺T cells were characterized as Treg cells, CD3⁺CD4⁺CD45RA⁺CD62L⁺ T cells were characterized as naïve T cells, and CD3⁺CD4⁺CD45RO⁺ T cells were characterized as memory T cells.

Apoptotic cell was identified using an apoptosis detection kit from BD Biosciences (USA). Huh-7 and Hep3B cells were incubated with either 0.01% DMSO (Control) or varying concentrations of flavokawain C (4, 8, and 16 μM) for 48 h. The cells were then stained with Annexin V and propidium iodide (PI) for 15 min. After that, Annexin V binding buffer was added to the mixture, and fluorescence was determined using a FACSC flow cytometer (BD Biosciences). The obtained data was analyzed with Flowjo 9.0 software.