Cell death was assessed by using a flow cytometer (BD Biosciences) and FlowJo software (Tree Star). The CD4+ T cells were isolated and activated, as previously described [12]. In brief, after differentiating DCs with or without ginsenoside fraction treatment, the cells were stimulated for 2 d with ethanol-killed Staphylococcus aureus (107 colony-forming units (CFU)/mL) [12]. After washing with PBS, 2 × 105 cells were cocultured in a 96-well plate with CD4+ T cells (2 × 105 cells) labeled with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Carlsbad, NM, USA). After 5 d, the cells were harvested and washed with PBS. The intensity of CFSE was determined by flow cytometry. Androgen Receptor Antagonist nmr After

culturing for 3 d, the IFN-γ levels in the supernatants were determined using an ELISA kit (R&D Systems). Comparative data were analyzed by the Student t test using the SAS statistical software package, version 9.3 (SAS Institute Inc., Cary, NC, USA). Differences were considered statistically significant when p < 0.05. We initially examined the proportion of each ginsenoside fraction in the sample by using TLC, which is a common

technique for the fingerprint analysis of a mixed complex because of its ease of use, low cost, and versatility. As Fig. 1A shows, Rg3, Rd, and Rb1 were the predominant components. We then examined the ginsenoside fraction further by using high performance liquid chromatography. As expected from TLC results, Rb1, Rg3, and Rd were the major components in the ginseng root, and Selleck MK-1775 the largest fraction was Rc (Fig. 1B). First, to examine the cytotoxicity of the ginsenoside fractions on CD14+ monocytes, we analyzed apoptosis of

CD14+ monocytes by using Annexin V/PI GNA12 for the first 5 d of differentiation. The ginsenoside fractions did not show any major signs of inducing apoptosis (Fig. 2A and B). These results suggested that 1 μg/mL or 10 μg/mL of ginsenoside fractions was a valid concentration to use for further experiments during DC differentiation. Second, to determine the effect of ginsenoside fractions on cytokine responses of CD14+ monocytes, the cells were treated for 24 h with ginsenoside fractions at a concentration of 0 μg/mL, 1 μg/mL, or 10 μg/mL. The supernatant was examined for cytokine production. As Fig. 3A shows, the expression of TNF-α (p < 0.001) and IL-6 (p < 0.01) increased significantly after treatment with ginsenoside fractions (at the concentration of 10 μg/mL), but IL-1β showed minimal changes. As Fig. 3B shows, IL-10 interestingly also increased in a dose-dependent manner. To confirm whether the induction of cytokines was because the ginsenoside fractions were contaminated with LPS, an LPS neutralization assay was performed, after the addition of PMB, which inhibits the LPS response [13]. As expected, the production of TNF-α in LPS-treated cells decreased significantly (p = 0.