Fluostar optima fluorecence plate reader

The primary cultured pituitary cells from prepubertal grass carp were incubated with several drugs, respectively. Then the culture medium was collected for measurement of SLα release using fluorescence-based ELISA. In this study, the recombinant grass carp SLα protein was synthesized and used to product the polyclonal antibody. Then the recombinant SLα protein was biotinylated and used as the tracer for the respective assays. Protein A (0.5 μg/mL) precoated Costar 96-well black plate (Thermo Fisher, MA) was used to load the protein samples, biotinylated SLα (12.5 ng/mL), and SLα antibody (0.45 μg/mL) (For information of SLα antibody, please refer to Supplemental Table S2). After overnight incubation at 4 °C, washing buffer was used to rinse each well to remove non-specific binding of the primary antibody. Then we introduced HRP-conjugate streptavidin (0.5 μg/mL) to incubate each well for anther 1 h at room temperature. After that, unbound second antibody was removed by decanting and washing three times. Then a 100 μL volume of QuantaBlu^TM Fluorogenic Peroxidase Substrate (Thermo Scientific, Rockford, USA) was then added into each well for signal development. Finally, FluoStar OPTIMA-Fluorecence plate reader (BMG Labtech GmbH, Ortenberg, Germany) was used to detect the fluorescence signals.

The grass carp pituitary cells were incubated with EGF. Then, after drug treatment, the culture medium was harvested for measurement of PRL, GH and LH release by using Fluorescence Immunoassay (FIA), respectively. In this experiment, the recombinant grass carp PRL, GH and LH protein were synthesized and used to produce the polyclonal antibodies, respectively. The recombinant proteins were biotinylated and used as the tracer for the respective assays. Costar 96-well black plate (Thermo Fisher, Waltham, MA, USA) was precoated with protein A (0.5 µg/mL), which was used to load the protein samples with tracer and antibody, respectively (For information of PRL, GH and LH antibody, please refer to Table S3). After overnight incubation at 4 °C, each well was rinsed three times with washing buffer to clear non-specific binding of primary antibody. Then, the plate was continued to incubate for another 1 h with HRP-conjugate streptavidin (0.5 µg/mL) at 26 ± 2 °C. Subsequently, unbound second antibody was eliminated by decanting. Then, QuantaBlu^TM Fluorogenic Peroxidase Substrate (Thermo Scientific, Rockford, IL, USA) was added into individual wells for signal development. Finally, fluorescence signals were routinely detected by using a FluoStar OPTIMA-Fluorecence plate reader (BMG Labtech GmbH, Ortenberg, Germany).