Anti-Inflammatory Effects of Ursolic Acid 24 h, harvested, fixed, permeablized and stained with PE labelled Bcl-2 antibody or Bcl-xl antibody. Representative flowcytometric histograms (E) and the corresponding bar diagram (F) are shown. Each bar shows mean6S.E.M from three replicates and two such independent experiments were carried out. *p,0.01, as compared to vehicle treated cells and #p,0.01, as compared to Con A stimulated cells. doi:10.1371/journal.pone.0031318.g006 Materials and Methods Chemicals Ursolic acid, RPMI-1640, HEPES, EDTA, EGTA, PMSF, leupeptin, aprotinin, benzamidine, dithiothreitol (DTT), glutathi- one (GSH), N-acetyl cysteine (NAC), NP40, propidium iodide, lipopolysaccharide (LPS) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (USA). Fetal calf serum (FCS) was obtained from GIBCO BRL. Concanavalin A (Con A) and trolox were purchased from Calbiochem, USA. ELISA sets for detection of cytokines (IL-2, IL-4, IL-6, IFN-c, TNF-a and IL- 1b) and monoclonal antibodies against Bcl-2, Bcl-xl, CD25, CD69, CD19, CD80, CD86, MHCII, CD134 and CD28 labeled with PE were procured from BD Pharmingen (USA). Antibodies against p-ERK, ERK, IkB-a, p-MEK, p-c-Raf, p-JNK, JNK and b-actin were obtained from Cell Signaling Technologies (USA). Approval of animal ethics Committee ‘The Institutional Animal Ethics Committee of Bhabha Atomic Research Centre, Government of India’, has approved the animal studies and the guidelines issued by the ethics committee regarding the maintenance and dissections of small animals were strictly followed. Project No. BAEC/11/10 and Date of approval: April, 2010. Treatment with ursolic acid A 20 mM solution of ursolic acid was prepared in dimethyl sulfoxide, stored as small aliquots at 220uC and diluted as needed in cell culture medium. In all in vitro experiments, cells were treated with different doses of ursolic acid for 4 hours before the initiation of culture. DMSO (0.1%) was used as vehicle control in vitro. Proliferation assay Splenic lymphocytes were obtained by squeezing the spleen through a nylon mesh in a petri plate containing RPMI medium. The RBC were lysed by brief hypotonic shock. Splenic lymphocytes were stained with CFSE (20 mM, 5 min, 37uC) and washed three times using ice-cold RPMI medium containing 10% FCS, 100 IU/ml penicillin and 100 mg/ml streptomycin. Two million splenic lymphocytes were treated with ursolic acid (0.25 mM to 5 mM, 4 h) and were stimulated with Con A (5 mg/ ml) or LPS (50 mg/ml) for 72 h at 37uC in 2 ml RPMI with 10% FCS in a 95% air/5% CO2 atmosphere. Vehicle treated cells served as a control. Cell proliferation was measured by dye dilution in a flowcytometer (Partec CyFlow). Percent daughter cells that showed a decrease in CFSE fluorescence intensity were calculated using FlowmaxH software and were expressed as daughter cells [50]. CD4+ and CD8+ T cell isolation and proliferation assay CD4+ (purity: 93%) & CD8+ (purity: 92%) T cells were isolated by using EasySep immunomagnetic cell sorting kit from Stem Cell Technologies, with PE labelled anti-CD4 antibody conjugated to magnetic nanoparticles through dextran and separation using magnetic field. For cell proliferation analysis, total splenocytes were first labelled with CFSE and then sorted and cultured for 24 or 72 h respectively for cytokine or proliferation assay. Estimation of cell cycle and apoptosis The percentage of cells in different phases of cell cycle (G1, S+G2/M) and percentage of apoptotic cells was estimated using a flowcytometer. One million splenic lymphocytes were treated with ursolic acid (0.25–5 mM) for 4 h and stimulated with Con A for 72 h at 37uC in RPMI1640 medium supplemented with 10% FCS in a 5% CO2 atmosphere. Vehicle treated cells served as control. The cells were washed with PBS and incubated with 1 ml of staining solution containing 0.5 mg/ml propidium iodide, 0.1% sodium citrate and 0.1% triton X-100 overnight [51]. A total of 20,000 cells were acquired on Partec Cyflow flowcytometer and analyzed using FloMaxH software. Undivided cells were in G1 phase of cell cycle (2n DNA content). The pre G1 population represented the apoptotic cells. The population showing more than 2n DNA represented cells in S+G2/M phase of cell cycle. Measurement of cytokine secretion The concentration of IL-2, IL-4, IL-6 and IFN-c in the supernatant of control unstimulated cells and cells stimulated with Con A for 24 h after ursolic acid treatment (4 h) was estimated using cytokine ELISA sets (BD Pharmingen, USA). The supernatant obtained from Con A stimulated cells was used as positive control. Cytokines induced by LPS was estimated in the culture supernatant of splenic adherent macrophage. Spleen cells (56106 cells/well) were incubated in a 24-well cell culture plate for 3 h at 37uC in a humidified atmosphere of 5% CO2 and 95% air. The non-adherent cells were removed by aspiration. The adherent cells (macrophages) were incubated with ursolic acid (5 mM for 4 h) and then stimulated with LPS (50 mg/ml) and further cultured for 6 h or 24 h at 37uC. The concentration of IL-6 and TNF-a in the supernatant of LPS stimulated cells for 6 h and IL-1b for 24 h was estimated using cytokine ELISA sets (BD Pharmingen, USA) [52]. Intracellular ROS measurements: To detect intracellular ROS, lymphocytes were incubated with 20 mM oxidation-sensitive dichlorofluorescein diacetate (DCF-DA) for 20 min at 37uC before being treated with various concentrations of ursolic acid. After 1 h of incubation, the increase in fluorescence resulting from oxidation of H2DCF to DCF was measured using a spectrofluorimeter [53]. Intracellular GSH assay To measure intracellular GSH, lymphocytes were treated with ursolic acid for 4 h at 37uC. Monochlorobimane (final concentra- tion, 40 mM, 30 min 37uC) was loaded into cells. Fluorescence emission from cellular sulfhydryl-reacted monochlorobimane was measured using a spectrofluorimeter (BMG Labtech Optima). Monochlorobimane is also known to react with small-molecular- weight thiols other than GSH but GSH forms the major monochlorobimane reactive thiol. Hence, MCB fluorescence is referred to as GSH levels in this manuscript. There are several reports in the literature measuring GSH levels using this dye. Intracellular and surface antibody staining Three million lymphocytes cells were cultured in presence or absence of ursolic acid for 4 h and then stimulated with Con A for 24 h at 37uC. Cultured cells were fixed with 4% paraformalde- hyde for 10 min at room temp and excess of paraformaldehyde was removed by washing once with wash buffer (PBS containing PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e31318