Mouse tgf β1 duoset elisa development system

CD4⁺T cells were purified from splenic single cell suspensions using FITC-conjugated monoclonal antibody to CD4 molecule (GK 1.5) and anti-FITC microbeads (Miltenyi Biotec K.K., Tokyo, Japan). Purified CD4⁺T cells (2.5 × 10⁵/200 µl) were stimulated with 5 µg/mL anti-CD3 + 2.5 µg/mL anti-CD28 in the presence of GolgiPlug and GolgiStop (BD PharMingen Co. Ltd.) for 5 h. Cells were fixed and permeabilized with the Cytofix/Cytoperm Plus kit (BD Biosciences Co. Ltd.) according to the manufacturer’s directions. Cells were stained with FITC or PE-conjugated monoclonal Ab against each cytokine. Purified CD4⁺T cells (2.5 × 10⁵/200 µl) were also stimulated with 5 µg/mL anti-CD3⁺2.5 µg/mL anti-CD28 for 48 h, and culture supernatants were analyzed for cytokine production using the Mouse Cytokine ELISA Plate Array (Signosis, Inc., Santa Clara, CA) according to the manufacturer’s directions. Samples in 96-well microtiter plates were analyzed on a LAS3000 (FujiFilm Ltd., Tokyo, Japan) and the chemiluminescent intensity of each sample was evaluated by Image Gauge ver. 4.0 (FUJIFILM Software, Yokohama, Japan). The ratios of each sample to a blank of intensity per mm³ were obtained. The production of TGF-β was analyzed with a mouse TGF-β1 DuoSet ELISA Development system (R&D Systems, Inc., Minneapolis, MN).

The level of TGFβ in supernatants of cultured YAMC cells was measured using the mouse TGFβ1 DuoSet ELISA development system (DY1679; R&D System, Minneapolis, MN), according to the manufacturer’s instruction. Purified TGFβ was used to generate the standard concentration curve. Cell numbers were counted at the end of experiments. The TGFβ concentration was representative as of ng/10⁶ cells.