Duoset elisa ancillary reagent kit 1

ELISA was performed using the cell culture supernatant collected at 48 h after siRNA transfection to minimize variation due to the increased number of dead cells in the siRNA-treated samples. We collected the cell culture supernatant and centrifuged at 400× g for 5 min at 4 °C to remove the floating cells. We harvested the supernatant and centrifuged again at 10,000× g for 10 min at 4 °C to remove the debris. The supernatant was frozen at −80 °C until the analysis. TGF-β1 ELISA with the activation of latent TGF-β1 was performed using Human TGF-β1 DuoSet ELISA (R&D Systems DY240-05) with DuoSet ELISA Ancillary Reagent Kit 1 (R&D Systems DY007) according to the protocol recommended by the manufacturer. We subtracted the TGF-β1 level with the level of medium from no-cell control wells, treated with mock transfection. Then, the level of TGF-β1 was corrected with the number of living cells and normalized to siLuc.