Spark multimode microplate reader

Samples were homogenized using liquid nitrogen and resuspended in 1.5 mL of tricine buffer (0.2 M pH 8). The extracts were incubated overnight at 4 °C, then centrifuged at 12,000× g at 4 °C for 10 min and the supernatants were recovered and filtered (0.45 μm pore size, Millipore) and stored at −20 °C until analysis.
The quantification of calcium was performed using the Calcium Assay Colorimetric Kit (Abcam, Cambridge, UK—ab272527; www.abcam.com/ab272527 (accessed on 15 December 2022)) and a microplate reader (Spark^® Multimode Microplate Reader—Tecan, Switzerland). Data are expressed as μg Ca²⁺ ⋅ mg f.w.⁻¹.
The TGase activity was monitored, using the Fluorescent Transglutaminase Assay Kit (T036—Zedira GmbH, Darmstadt, Germany) and a microplate reader (Spark^® Multimode Microplate Reader—Tecan, Switzerland) by measuring the fluorescence (excitation wavelength 332 nm; emission wavelength 500 nm). The relative TGase activity was detected by the increasing of fluorescence intensity over time. Enzyme activity is expressed as the %, compared to the untreated control.

DENV-R2A/ZIKV-R2A-Infected cells were lysed in 100 μL lysis buffer (0.1% Triton X-100, 25 mM glycylglycine [pH 7.8], 15 mM MgSO₄, 4 mM EGTA [pH 8], and 1 mM DTT). RLuc assay was performed by mixing 30 μL of the lysate with 150 μL of assay buffer (25 mM glycylglycine [pH 7.8], 15 mM MgSO₄, 4 mM EGTA [pH 8], 15 mM K2PO₄ [pH 7.8]) and coelenterazine (1.43 μM, Prolume). Luminescence was measured using Spark multimode microplate reader (Tecan).
To assess the efficiency of drugs on HCV, Huh7.5 cells were washed twice in PBS, resuspended in Ingeno buffer (Mirus) at 1 × 10⁷ cells/mL. Electroporation was performed in 0.2 cm cuvette with 2 × 10⁶ cells using the following parameter: 820 V, 99 μs, 4 pulses, 1.1 s interval with an ECM 830 Electro Square Porator (BTX). Electroporated cells were seeded in 24-well plates with 1.5 × 10⁵/well. Four hours later, medium was changed to complete DMEM media containing NPP3C or 6A1HI at 50 μM. DMSO and BILN2061, a HCV NS3 protease inhibitor (a kind gift from Daniel Lamarre, University of Montreal), were used as negative and positive control, respectively. Cells were lysed and tested for luciferase 48 h posttransfection using 100 μL of BrightGlo reagent (Promega). Firefly luciferase activity was measured with a Spark multimode microplate reader (Tecan). NPP3C, 6A1HI, and their 151 analogues were obtained from Key Organics.

A sterile 55% w/v royal jelly solution was diluted in brain heart infusion (BHI; Himedia Laboratories) broth to obtain a concentration of 50% and then serial dilution was performed in 96 well microplates. A total of 5 µl of the
P. aeruginosa ATCC 10145 suspension or clinical isolate bacteria (1.5 × 10
⁵ CFU/ml) was inoculated in all groups, except the groups that had been determined as blanks. The culture was then incubated at 37°C for 18 hours. After that, the microplate was scanned using the Spark® Multimode Microplate Reader (Tecan trading AG) to measure optical density (OD) using a 600 nm wavelength. The percentage of bacterial viability inhibition was determined based on the OD value of the treatment group against the control. The bacterial viability assay experiments were carried out in quadruplicate.