Human survivin quantikine elisa kit

To determine the release of survivin, human connective tissue growth factor (CTGF) and extracellular signal-regulated kinase 1 (ERK1) enzyme-linked immunosorbent assays (ELISA) were used. 1 × 10⁴ MCF10A or 1 × 10⁴ MCF-7 cells were seeded into each well of a 12-well plate. 1 μM of SVA was added 24 h later. At the end point of 48 h after irradiation, culture medium samples were collected, centrifuged, shock-frozen by means of nitrogen and stored at −80 °C until assayed. Simultaneously, the cells were detached by 0.05% trypsin/EDTA solution and centrifuged at 250 x g. Cell pellets were washed twice with PBS and lysed at 1 × 10⁷ cells/ml Lysis Buffer (R & D Systems, USA) on ice. The concentration of survivin and CTGF in the cell culture supernatant was assayed using the Human Survivin Quantikine ELISA Kit and the Human/CCN2 DuoSet ELISA (R & D Systems). The content of ERK1 in cell lysates was determined using the Human Total ERK1 DuoSet IC ELISA (R&D Systems). Optical densities were read using Anthos Zenyth 340 Plate Reader. The respective standard curves and protein concentrations were calculated via “Four Parameter Logistic Curve” online data analysis tool, MyAssays Ltd., 24 th. November 2012, http://www.myassays.com/four-parameter-logistic-curve.assay.