Biotek cytation 5 system

Following the exposure of embryos/larvae at 120 hpf, TH levels were measured from the homogenates of pooled fish (n = 200/replicate) using an enzyme-linked immunosorbent assay (ELISA) according to the protocol described by Yu et al. (2010) [22 (link)] with minor modifications. Briefly, 200 zebrafish larvae in a glass beaker were homogenized in 200 µL of 1× PBS using a pestle tissue grinder. The samples were then centrifuged for 10 min at 5000 × g and 4 °C. The supernatant of the samples was collected and stored at −80 °C until further measurement. The levels of hormones were quantified using ELISA kits (Cusabio Biotech, Wuhan, China) for T4 (Cat no. CSB-E08489f), T3 (Cat no. CSB-E08488f), and TSHβ (Cat no. CSB-EQ027261FI) with a BioTek Cytation 5 system (BioTek, Winooski, VT, USA) according to the manufacturer’s recommended protocol. The optical density for T4, T3, and TSHβ was determined using BioTek Cytation 5 system (BioTek, Winooski, VT, USA) set to 450 nm. The limits of detection for T4, T3, and TSHβ are reportedly 20 ng/mL, 0.5 ng/mL, and 2.5 µIU/mL, respectively. The measurements of T4, T3, and TSHβ were normalized to the protein level (mg/mL). Protein levels were analyzed using a BCA protein assay kit (Thermo Fisher Scientific, Johannesburg, South Africa) and BioTek citation 5 (BioTek, Winooski, VT, USA) system set to 562 nm.

For quantification of 680RD-labelled leptin level in tissue, the Biotek Cytation 5 System (BioTek) was used. Tumour and skin tissues were snap-frozen in liquid nitrogen and sonicated in Lysis Buffer (R&D Systems) before centrifuging at full speed at 4 °C for 10 min to obtain total lysates. Serial dilutions of labelled recombinant protein were used as standards to generate a curve to estimate the amount of protein in the tissue lysates. A total of 100 μl of lysates and standards in duplicates were loaded into 96-well black assay plate (Thermo Fisher Scientific) and then read at an excitation of 680 nm and emission of 695 nm. Estimated concentrations were then calculated.

Cell viability of BMSCs were tested using the CCK-8 (Dojindo Molecular Technology, Kumamoto, Japan) according to instruction. BMSCs were inoculated in a 96-well plate (5 × 10³ cells per well) and hatched overnight. On the second day, cells were treated with PTH (1-34)/PTHG2 with different concentrations (0, 0.005, 0.01, 0.1, 1, and 100 μM) for 48 h and 96 h. Then, cells were disposed with 10 μl of CCK-8 reagent for 2 hours. After incubation, the absorbance was measured at 450 nm in a Biotek Cytation 5 System (Biotek Instruments, Vermont, USA).