Fresh green tea extract   Whole green tea (Camelia sinensis L) ex

Fresh green tea extract.  Whole green tea (Camelia sinensis L) extract (Topix Pharmaceuticals, West Babylon, NY, USA) was suspended in RPMI-1640 (Sigma, St. Louis, MO, USA) at a concentration of 1 g/100 ml and further diluted for the experiments. The extract contained a 90% polyphenol isolate from whole leaf, with 80% catechins; EGCG composed 70% of catechins. GTE was freshly prepared prior to each experiment, and leftover solution was stored PI3K inhibitor at 4°C. Epigallocatechin Gallate.  Purified EGCG (>95% purity; Sigma-Aldrich, St. Louis, MO, USA) was suspended in RPMI-1640

(Sigma) at a concentration of 1 g/100 ml and further diluted to concentrations of 50% because of the 50% content of EGCG in the GTE used. The GTE contained 90% polyphenols, and 80% of the polyphenols are catechins. 70% of the catechins are EGCG, which approximates to 50% of the GTE is EGCG. Based on the above, the EGCG concentration in culture was 50% of the GTE concentration. Cell Cultures.  Human PBMC (1.5 × 106 cells/ml) were separated on a Ficoll-Paque (Pharmacia, Piscataway, NJ, USA) gradient (density 1.077) and washed twice in RPMI-1640 medium (Gibco/BRL, Grand Island, NY, USA) and counted. Cells check details were then cultured in complete RPMI medium (c-RPMI) containing L-glutamine (2 mm) (Sigma), penicillin (100 Units/ml)

(Sigma), streptomycin (100 μg/ml) (Sigma) and N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid buffer (HEPES) (25 mm) click here (Sigma) and supplemented with heat-inactivated foetal calf serum (FCS) (10%) (Gibco), ± recombinant human interleukin-4 (IL-4) (100 ng/ml) (R&D), ± mouse anti-human monoclonal (mAb) CD40 (1 μg/ml) (BD Pharmingen Transduction Labs, San Diego, CA, USA), ± varying concentrations of GTE (1–100 ng/ml) (Topix Pharmaceuticals, West Babylon, NY, USA) or EGCG (0.5–50 ng/ml) (Sigma). In some experiments,

cat pelt antigen (1 AU/ml) (Alk-Abelló, Hørsholm, Denmark) was added to cultures to assess for differences between allergen- and non-specific IgE responses; cat pelt was chosen because all three subjects had positive SPT to cat pelt. Control cultures included anti-CD40 and rhIL-4 without cat pelt antigen. The cells were then cultured for 10 days at 37°C in a humidified atmosphere of 4% CO2 in air, after which supernatants were collected and frozen (−20°C), and then assayed for IgE production. (ELISA, BioQuant, San Diego CA, USA). Cell viability.  Cell viability was >90% as judged by trypan blue (Gibco) exclusion on day 10 in all cultures (±GTE). Quantification of IgE production.  In vitro quantitative determination of IgE content in cell culture supernatants was performed using a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) (IgE ELISA Test Kits, BIOQUANT). All ELISAs were performed according to the manufacturer’s recommended procedure. Specimens were analysed in triplicate and a standard curve was derived from known concentrations of IgE.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>