Automated microplate reader

Mitochondrial function was determined in intact cells by real-time respirometry (Seahorse XFe24; Agilent). MDA-MB-231 and EO771 cells were seeded at 1.0 × 10⁴ cells/well in an XFe 24-well plate (Agilent). Once cells reached 80% confluency, they were treated with a vehicle (0.01% DMSO) or varying concentrations of BAM15 for 16 h. Following treatment, media was removed, and cells were incubated at 37°C for 1 h in XF DMEM medium (pH 7.4) supplemented with 1 mM pyruvate and 2 mM glutamine without CO₂. Cells were then serially injected with 10 mM glucose, 1 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone and antimycin A. Components of mitochondrial function were calculated as described previously [67 (link)]. Data were normalized to nuclear content by staining live cells after assay with 20 μM Hoechst 33342 (ThermoFisher Scientific) and reading fluorescence on an automated microplate reader (Biotek) at excitation/emission 350/461 nm. Mitochondrial respiration was quantified as described previously [18 (link)].

To measure the specific immunoglobulin Y (IgY) antibodies against NDV, the sera from blood samples were collected partially from chickens, via wing-web bleeding, at 20, 34, and 49 days post-vaccination (dpv). All serum samples were separated from blood samples by centrifugation at 3500 rpm for 7 min at room temperature. For the ELISA, the commercial kit ID Screen^® Newcastle Disease Indirect (ID.Vet, Grabels, France) was used following the manufacturer’s protocol. The reading was made at 450 nm using an automated microplate reader (BioTek Instruments Inc., Winooski, VT, USA). All serum samples were analyzed using a positive and negative control antisera provided by the kit, and the antibody titers were quantified using the software provided by the manufacturer. The serum samples with the ELISA antibody titer ≥ 993 were considered positive. The HI was only evaluated at 49 dpv; the antibody titers were calculated using 4 haemagglutination units (HU) of the LaSota strain as an antigen, following the procedure described by World Organization for Animal Health (OIE) Terrestrial Manual-Chapter 3.3.14. Newcastle Disease (Infection with Newcastle Disease Virus) [50 ].

To measure expressions of inflammatory mediators in hepatic tissues, liver (right lobe) was homogenized in ice-cold RIPA (radioimmunoprecipitation assay) buffer using a bead homogenizer (taco™Prep Bead Beater, GeneReach Biotechnology Corp., Taichung, Taiwan). After centrifugation at 20,000× g for 20 min, the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 in the resulting supernatant were measured by commercial enzyme-linked immunosorbent assay (ELSIA) kit (Mybiosource, San Diego, CA, USA), following manufacturer’s instructions.
In the case of in vitro experiment, nitric oxide (NO), prostaglandin E₂ (PGE₂), TNF-α, IL-1β, IL-6, and monocyte chemoattractant protein-1 (MCP-1) expressions in conditioned media were quantified. To measure NO, conditioned media (100 µL) was reacted with the same volume of Griess reagent, and absorbance at 540 nm was detected using an automated microplate reader (BioTek). Expressions of proinflammatory cytokines including TNF-α, IL-1β, IL-6, MCP-1 (BD Biosciences), and PGE₂ (R&D Systems, Minneapolis, MN, USA) in conditioned media were measured using commercial ELISA kit.

The transfected preadipocytes were seeded into 96-well plates. Then, 24 h after transfection, 10 μL of CCK8 reagent (Beyotime, Beijing, China) was added to each well, and the incubation was continued for 2 h at 37 °C. In addition, the cell proliferation index was assessed at 450 nm using an automated microplate reader (BioTek, Winooski, VT, USA) according to manufacturer’s instructions.

In vitro VEGFR2 tyrosine kinase activity was analysed by using an enzyme-linked immunosorbent assay kit as previously described [26 (link)]. Briefly, the assay in 96-well plates, employs an affinity tag labelled capture antibody and a reporter conjugated detector antibody which immunocaptures the analyte in solution. The complex (captured antibody/analyte/detector antibody) is, in turn, immobilised via immunoaffinity of an anti-tag antibody coating the well. Pd₂Spm and DTX were tested separately (at their IC₅₀ concentrations, 1.7 and 1.8x10^-2 μM, respectively), or in combination (at 6x10^-1/6x10^-3 μM for Pd₂Spm/DTX), by incubation with the antibody mixture. Colour development was determined at 450 nm, in an automated microplate reader (Biotek Winooski, USA).