Spark multiplate reader

GCase enzyme activity was measured through the fluorescent substrate 4-methylumbeliferyl-N-acetyl-β-glucosamine (Sigma-Aldrich M-3633) method. The test was performed on protein lysate, extracted through sonication in PBS + Triton X-100 0.01%. 1 mg protein was incubated with 100 μL of substrate and water or CBE as negative control. After 1 h at 37°, the fluorescence level was measured using the Spark multiplate reader (Tecan, Männedorf, Switzerland). The enzyme activity was expressed as nmol/mg/h.

All reagents were of analytical grade purity, and unless specified otherwise, all reagents were acquired from Sigma-Aldrich (Merck, Bucharest, Romania).
For the antioxidant capacity assays performed, the extracts were diluted to a concentration of 100 mg/mL in 30% ethanol solution. Rutin Equivalent Antioxidant Capacity (REAC), in fact ABTS bleaching assay with rutin as standard [79 (link)] and cupric ion reducing antioxidant capacity (CUPRAC) [79 (link)] are presented in the Supplementary Material.
Induced peroxidation of liposomes (LipPx) assay was performed as follows: Liposomes of 0.2 mg/mL concentration in phosphate buffer saline (PBS) of 7.4 pH were sonicated for 30 min until homogenization [80 (link)]. Further, 290 μL of this solution was mixed with 10 μL of each extract (10 μg/mL) and the reaction was triggered by the addition of cytochrome C, to a final concentration of 2 μM. The reaction evolution was recorded at 235 nm using the Tecan Spark multiplate reader overnight, at 25 °C. The corresponding lag phase was determined as the value of inflexion point for all kinetic measurements. Rutin (in 0.067–2.3 μg/mL range) was used as a standard to obtain the kinetic curve.

For the ADCC assay, Mia PaCa-2 and PL45 cells were seeded in a 96-well plate at the density of 5 × 10³ cells/well with DMEM supplemented with 10% FBS and then treated with Oba01 for 30 min. Next, the cells were incubated with human peripheral blood mononuclear cells (PBMCs) (target cells: effector cells ratio 1:5, 1:20, 1:80) for 4 h. For the CDC assay, the serum samples were first separated from the whole blood samples obtained from two healthy volunteers. A half portion of each sample was incubated in a 56 °C water bath for 30 min to inactivate the complement activity. Mia PaCa-2 and PL45 cells were seeded in a 96-well plate at the density of 5 × 10³ cells/well with the DMEM culture medium supplemented with 10% FBS and then treated with Oba01 for 30 min. Subsequently, the cells were incubated with the prepared human serum samples (final concentration 20%) for 4 h. After incubation, the cells were lysed with lysis buffer for 45 min, and the CytoTox 96^® Non-Radio Reagent (Promega) was added to measure the OD₄₉₀ values on the SPARK Multiplate Reader (TECAN, Switzerland), after which the lysis rate (%) was calculated.

NanoLuc concentrations in cell culture supernatants were quantified with the Nano-Glo Luciferase Assay System (N1110, Promega). 10 μL of cell culture supernatant was mixed with 10 μL of buffer/substrate mix (50:1) in 384-well plates (781076, Greiner Bio-One), which were briefly centrifuged at 1200 rpm and incubated for 5 min at room temperature. Luminescence intensity was measured using a Tecan Spark multiplate reader (Tecan AG).

For the real-time in cellulo fLuc activation kinetics experiment, D-luciferin (LUCK-100, Gold Biotechnology) was added to the iCROP-fLuc-transfected cells to the final concentration of 1.5 mM. Plates were incubated for 30 min at 37 °C before addition of the inducer and then the luminescence intensity increase was immediately measured every 10 s for a total duration of 8 min with a Tecan Spark multiplate reader (Tecan AG).

A panel of PADC cell lines was treated with a tenfold dilution series of Oba01 in triplicate in media supplemented with 10% heat-inactivated bovine serum (Gibco) for 3 days. The cell viability was measured with the cell Titer-Glo® assay kit (G7572, Promega, Madison, USA) in accordance with the manufacturer’s instruction, while the absorbance was determined by the SPARK Multiplate Reader (TECAN, Switzerland). The untreated cells served as a control. The cell survival rate (%) was calculated using the following formula: Asample/A control × 100%. The 50% inhibitory concentration (IC50) was calculated by nonlinear regression analysis (GraphPad Prism 5.0). The coefficient of drug interaction (CDI) was calculated to evaluate the interaction between two drugs, as reported previously [23 (link)].