Thermo multiskan ex

First, 96-well plates (Corning, VWR) were coated overnight at 4 °C with 0.5 μg/well fibronectin (Sigma). SK-Mel-2 cells were trypsinised, washed and resuspended in RPMI medium only at 10⁵ cells/mL. Cells (100 µL/well) were treated with compound (DMSO final concentration 0.1%) at the indicated concentration for 4 hrs on a rotary shaker. Plates were washed (3 × PBS) and blocked with PBS/5% BSA for 2 hrs at 37 °C prior to adding the cells. Cells were incubated on the plates at 37 °C in a humidified chamber with 5% CO₂ for 1 hr. The plates were washed 3× with PBS and a 200 µL/well RPMI medium containing 10% FCS added. The plates were incubated overnight at 37 °C as above. Finally, 0.5 mg/mL of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added to each well and the plates incubated for a further 4 h. The medium was removed, and the insoluble formazan dissolved in 150 µL of DMSO. Absorbance was measured at 540 nm using a Thermo Multiskan EX (Thermofisher, UK)and Ascent Software (v2.6). Total binding was determined based on controls lacking any compounds (100% binding) in fibronectin-coated wells and uncoated wells blocked with BSA (0% binding) and corrected for background (no cells, fibronectin-coated).

The yeast culture in the SDB medium was harvested by centrifugation (5,000×g, 5 min), washed, and resuspended with saline solution (0.85%) (19 (link), 23 (link)). The absorbance was measured at 0, 2, and 24 h in a spectrophotometer (Thermo Multiskan EX, Thermo Scientific, USA) at 600 nm without shaking the cell suspension. The autoaggregation was calculated as follows:
$\begin{array}{l}\text{Autoaggregation}(\text{\%})=(1-\frac{At}{A0})\times 100\end{array}$
A_t is the absorbance at 600 nm and A₀ is the initial absorbance at 600 nm.

Anti-CSE1L (H2) antibody-coated 96-well plates (Costar) were blocked with 5% BSA in TBS (Tris-buffered saline) for 1 h. The wells were washed with TBST (0.05% Tween 20 in TBS) and then incubated with serum samples (sixfold dilution with TBS) for 1 h. After being washed with TBST, the wells were incubated with biotin-conjugated anti-phospho-CSE1L antibodies for 1 h. The biotin-conjugated anti-phospho-CSE1L antibodies were prepared by biotinylating anti-phospho-CSE1L antibodies using the Biotin Labeling Kit-NH2 (Dojindo Laboratories, Kumamoto, Japan). The wells were then washed with TBST, reacted with streptavidin-conjugated horseradish peroxidase (R&D Systems, Minneapolis, MN, USA), and followed by incubation with the substrate reagent (R&D Systems). For calibration, two blank wells containing TBST were used as the background value, and two wells that were not coated with anti-phospho-CSE1L antibodies but reacted with all other ELISA reagents were used as control wells. The absorbance at 450 nm was measured within 20 min following the reaction by using a Thermo Multiskan EX microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was assayed two times.