Spectramax m2e spectrophotometer

Steady state kinetics of Eg5 were monitored using a NADH coupled enzymatic assay (46 (link), 71 (link)) in a SpectraMax M2e spectrophotometer (Molecular Devices) in 96-well plate format (47 (link)). In measurements of basal ATP hydrolysis rates in the presence of saturating 1 mm MgATP, motor protein concentration was as follows: L263F and T100C/L263F, 0.625 μm; T100C and Y82A, 1.5 μm; WT, Q78A, M115A, L263A, M115A/L263A, M115I/L263F, P137A, V298C, and P137A/V298C, 2.5 μm; and R138A, M115I, Q78N, Y82F, R138L, Q78A/R138A, and Y82F/T100C, 5 μm. Measurements of 12 ATP concentrations, ranging from 238 nm to 1 mm MgATP, were used to determine the dependence of kinesin catalysis on ATP concentration. For these reported k_cat and K_m experiments, 1242 assays were performed.

TNFα, IL-1ra, oncostatin M (OSM), OPN, and OC secretions were assessed from coculture supernatants by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Biomedical Technologies, Stoughton, MA, USA). TNFα and IL-1ra levels were determined 72 hours and 1, 2, and 3 weeks after seeding or the last IL-4 treatment, whichever was later. OSM secretions were similarly determined at 72 hours. Late osteogenic markers OC and OPN were analyzed 3 weeks after seeding. Coculture cell lysates were evaluated for ALP activity, an earlier osteogenic marker, using an ALP assay kit (BioAssay Systems, Hayward, CA, USA) 2 weeks after seeding. Manufacturers’ protocols were followed and colorimetric changes were measured with a SpectraMax M2e spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

Viability of primary astrocytes was assessed via an alamar blue assay (ThermoFisher, #DAL1025). Cells were seeded into PDL-coated black wall clear bottom 96-well plates (Greiner, #655090). Following experimental treatment, alamar blue was added (10% v/v) and incubated for 4 h at 37°C. Resazurin, the active compound of the assay, is metabolized to resorufin in living cells. Resazurin fluorescence was detected using SpectraMaxM2e spectrophotometer (Molecular Devices) using 570-nm excitation and 590-nm detection wavelengths. The signal was corrected for background fluorescence and the viability was calculated as a % control (untreated) cells.

Cell cytotoxicity was assessed via the measurement of lactate dehydrogenase leakage into the culture medium using a commercially available kit from Pierce (Thermo Scientific, UK, cat no. 88953). This was carried out in accordance with the kit guidelines, with the activity of LDH being calculated from the change in absorbance at 340 nm as NADH is reduced. HEK293 cells overexpressing mts17β-HSD10 were cultured in phenol-red free media (10% FBS, 1 mM Sodium Pyruvate, 100 units Penicillin, 0.1 mg/mL Streptomycin and 2 mM L-Glutamine) and seeded at a density of 10,000 cells per well (100 µL, 96-well plates). Cells were then treated with compound of interest at 2 concentrations (25 µM and 100 µM in DMSO) in triplicate. Treated cells were then incubated at 37 °C and CO₂ (5%) for 24 hours before the LDH assay was performed as per the manufacturer’s instructions. Spontaneous control (water) and maximum control (lysis buffer) used in accordance with the kit guide. Absorbance was measured at 490 nm and 680 nm using the SpectraMaxM2e spectrophotometer (Molecular Devices, San Jose, CA, USA). The measured LDH activity was used to calculate % cytotoxicity using the following equation: $\%\mathrm{cytotoxicity}=\frac{\left(\mathrm{compound}\mathrm{treated}\mathrm{LDH}\mathrm{Activity}-\mathrm{Spontaneous}\mathrm{LDH}\mathrm{Activity}\right)}{\left(\mathrm{Maximum}\mathrm{LDH}\mathrm{Activity}-\mathrm{Spontaneous}\mathrm{LDH}\mathrm{Activity}\right)}\times 100$

To determine the enzyme activity on chromogenic substrates, the absorbance was measured at 405 nm to estimate the release of p-nitrophenolate ions (pNP, ε405 = 0.0187 μM cm^-1, pH 10) following a reaction [40 (link)]. The initial reaction (100 μL) comprised 82 μL of 20 mM sodium phosphate buffer, pH 7, including 1 mM substrate (final concentration) and 18 μL of Bg10 at a concentration of 0.02 mg mL^-1. The addition of the enzyme to the reaction mix was timed at 1-min intervals. The reactions were incubated in a thermal cycler at 37°C or at the temperatures specified in the description of the respective experiments. Following incubation under the relevant assay conditions, 30 μL aliquots were added to 150 μL of sodium carbonate buffer (500 mM, pH 10) in 96-well, flat-bottomed, tissue culture plates (JetBiofil, Guangzhou, China) and homogenised by pipetting with a semi-automatic multichannel pipette. The released pNP was immediately measured at 405 nm using a SpectraMax^® M2^e spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). All assays were performed in triplicate, and the following controls were used: reactions without enzyme and reactions without the assayed compound. The enzyme activity was expressed in percentages relative to the highest value or to the specified standard for each essay.

Total protein extract from trypomastigotes (Dm28c-Luc; 10⁸ parasites/mL) were extracted with lysis buffer [13 (link)]. Cysteine protease activity of extracts (5 μg) was measured in acetate buffer using fluorogenic peptide substrate (60 μM of 7-Amino-4-Methylcoumarin hydrochloride, CBZ-L-Phenylalanyl-l-Arginine amide (Z-FR-AMC) [13 (link)]. Total protein extracts were incubated for 45 min at room temperature with the compounds and changes in relative fluorescence units monitored using a SpectraMaxM2e spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) for 1 h with excitation at 370 nm and emission at 460 nm. The inhibition assays were performed by coincubation with different compounds (3g, 3j, and 3m) and concentrations (300–11.11 µM) and transepoxysuccinyl-l-leucylamido-(4-guanidino) butane (E-64). Additional control was carried out with DMSO (≤1%). Enzymatic activity was expressed in µmol/min/mg of protein and the residual activity in percentage.