Biotek synergy lx

In vitro cytotoxicity of the Fab was determined by the WST-1 tetrazolium salt colorimetric growth assay. NT2-D1 cells were trypsinized, counted, and seeded at a density of 3 × 10³ cells/well on a 96-well plate overnight. The next day, cells were treated with anti-Nodal antibodies at concentrations ranging between 1 and 100 nM (commercial anti-Nodal WS65, positive control) and between 10 nM and 10 µM with the rhFab_3D1. After 72 h, cell viability was estimated adding 1/10 volume of WST-1 solution (Roche, Basel, Switzerland) and incubating for 1 h at 37 °C. The absorbance at 450/650 nm was determined using a microplate reader (BioTek Synergy LX, Agilent, Cernusco sul Naviglio, Italy). Results were expressed as percentage of control. Experiments were performed in three replicate wells and at least thrice. Data are reported as the mean of all experiments with associated standard error values. Curves were fitted using nonlinear regression using Graph Pad version 9.2.

Biochemical and hormonal assessments were performed on the serum samples from the following groups: CTR (n = 8), WD (n = 8), CS (n = 6), and WD/CS (n = 6). The serum glucose and lipid profiles were measured using a Mindray BS-200 Chemistry Analyzer (Shenzhen Mindray Bio-Medical Electronics Co., Shenzhen, China). The level of serum 17beta-estradiol was determined with a chemiluminescent immunometric assay on an IMMULITE 2000 analyzer (Siemens Healthcare Diagnostics, Eschborn, Germany). The serum concentrations of Insulin and Leptin were measured using commercial ELISA kits according to the manufacturer’s instructions (Leptin: Thermo Fisher Scientific, Waltham, MA, USA, intra-assay coefficient of variation (%CV): 4.3 %, and the sensitivity of the assay was 22 pg/mL; Insulin: R & D Systems, Minneapolis, MN, USA, % CV: <10%, the sensitivity was 5 μlU/mL). The absorbance at 450 nm was measured using a microplate reader (Biotek Synergy LX, Agilent, Santa Clara, CA, USA).
Insulin sensitivity was calculated in the serum samples from the rats by means of the quantitative Insulin sensitivity index (QUICKY = 1/(log(I) + log(G)), where I is the fasting serum Insulin concentration, and G is the fasting glucose concentration [27 (link)].

Culture supernatants (CS-PLGA nanoparticles (0.1 mg/mL) with surface-bound β-glucan at 0, 5, 10, 15, 20, or 25 ng or free β-glucan at 5, 10, 15, 20, or 25 ng/mL) were analyzed for TNFα using an ELISA kit according to the manufacturer’s instructions (KHC3011, Thermo-Fisher Scientific) (TNFα Human ELISA Kit–Thermo-Fisher Scientific). Briefly, a standard curve in the range of 0 pg/mL to 1,000 pg/mL of recombinant human (hu) TNFα was generated. 100 μL of standards or unknowns were added to each well and incubated for 2 h at room temperature. The plates were washed and 100 μL of Hu TNFα Biotin Conjugate solution was added to each well and incubated for 1 h at room temperature. The plate was washed and the 100 μL 1X Streptavidin-HRP solution was added to each well and incubated for 30 min at room temperature. The plates were washed and 100 μL stabilized chromogen was added to each well and incubated for 30 min at room temperature in the dark. 100 μL of Stop Solution was added to each well. The plates were read at 450 nm using a BioTeK Synergy LX multimode reader (BioTek-Agilent). The concentrations of each sample were detected based on optical density and the concentration of the standard.