System 2 software

After viability determination of harvested lymphocytes from each group using 0.04% trypan blue (viability > 90%) and adjusted cell concentration to 1 × 10⁶ cells/ml in PBS containing 2% FBS, the cells were incubated with surface markers including phycoerythrin (PE)-labeled anti-mouse CD3, Allophycocyanin (APC)-labeled anti-mouse CD4 and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 (eBioscience) at 4°C for 30 min in the dark. Followed by washed by 2 ml PBS and then fixed with FACScan buffer (PBS containing 1% FCS and 0.1% Sodium azide) and 2% paraformaldehyde, the cultures were analyzed of fluorescence profiles on a FACScan flow cytometer (BD Bio-sciences) by SYSTEM II software (Coulter).

The concentration of the purified splenocytes were adjusted into 1× 10⁵ cells/mL. Then the phycoerythrin (PE)-labeled anti-mouse CD3 (eBioscience) (5 μg/mL), Allophycocyanin (APC)-labeled anti-mouse CD4 (eBioscience) (5 μg/mL) and fluorescein isothiocyanate (FITC)-labeled anti-mouse CD8 (eBioscience) (5 μg/mL) antibodies were used to stain the T cell subclasses (CD4+ and CD8+) for 30 min at 4 °C. After washing by PBS, the cultures were fixed with FACScan buffer (1% FCS and 0.1% Sodium azide in PBS) and 2% paraformaldehyde. The T cell subclasses were measured for fluorescence profiles on a FACScan flow cytometer (BD Bio-sciences) and analyzed by SYSTEM II software (Coulter).

Spleens of three mice each group were aseptically removed and the single-cell suspensions were prepared for lymphocyte proliferation assay as previously described (23 (link)). Briefly, the cells were re-suspended in RPMI 1640 medium (Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), followed by counted with 0.04% trypan blue (Bio-Rad, California, USA), plated in 96-well costar plates (5 × 10⁵ cells/well) and incubated with TLA (10 μg/ml), concanavalin A (ConA, 5 μg/ml, Sigma, Missouri, USA) or medium alone at 37 ^oC in an atmosphere of 5% CO₂/air. Lymphocyte proliferation evaluated by stimulation index (SI) was determined by 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml, Sigma). SI was calculated as the ratio of OD₅₇₀ value of wells containing stimulus (TLA or ConA) compared with the medium (OD_570M).
For flow cytometry assay, 1 × 10⁵ cells were incubated with PE anti-mouse CD3e, APC anti-mouse CD4 and/or FITC anti-mouse CD8a (eBioscience, San Diego, USA) at 4 °C for 30 min in dark followed by washed with 2 ml of PBS (pH 7.4), fixed by FACScan buffer and blocked with 2% paraformaldehyde (17 (link)). Data was collected using a FACScan flow cytometer (BD Bio-sciences, USA) and the SYSTEM II software (Coulter).