Infinite 200 pro multiplate reader

Cells were seeded in 96-well plates and incubated overnight at 37°C with 5% CO₂. Upon treatment with respective inhibitors for 3 hrs or siRNAs for 48 hrs, 10μL of alamarBlue reagent (Invitrogen) was added to each well, before incubation at 37°C with 5% CO₂ for 4 hrs. Fluorescence was detected at an excitation wavelength of 570 nm and an emission wavelength of 585 nm on an Infinite 200 Pro multiplate reader (Tecan). Cell viability was analysed by normalizing to untreated cells [26 (link)].

HUH 7 cells were seeded on a 96-well plate and incubated at 37 °C with 5% CO₂ overnight. The cells were then treated with varying concentrations of catechin and incubated for 48 h. After incubation, media in the wells were replaced with alamarBlue™ (Thermo Fisher Scientific, Waltham, MA, USA) diluted in DMEM, supplemented with 2% FCS at a 1:10 dilution, and the 96-well plate was incubated for another 2.5 h at 37 °C, 5% CO₂. Infinite 200 Pro multiplate reader (Tecan, Männedorf, Zürich, Switzerland) was used to obtain fluorescence readings at an excitation wavelength of 570 nm and an emission wavelength of 585 nm. Relative cell viability was evaluated through normalizing the fluorescence readings of catechin-treated cells to 0.1% DMSO-treated cells.
Assays of dose-dependent inhibition were carried out to determine the inhibitory effects of catechin on virus replication. HUH 7 cells were seeded, left overnight, and subsequently infected with DENV for 1 h at 37 °C, 5% CO₂. For CHIKV, the infection duration was 1.5 h. Following infection, cells were washed one time with 1× PBS and exposed to 25 µM, 50 µM, or 100 µM catechin suitably diluted in DMEM with 2% FCS, or RPMI with 2% FCS, and incubated at 37 °C, 5% CO₂ for 48 h. The supernatants were harvested following incubation and viral plaque assays were performed to quantify viral titres.

MTS assay as an approved cytotoxicity test was performed to determine the non-toxic concentrations of both ribavirin and cisplatin on Vero cells using an MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) kit (Promega, USA) according to the manufacturer’s protocol. In short, the Vero cells were grown in 96-well plates and were treated with increasing concentrations of ribavirin and cisplatin in triplicate. The cytotoxicity assay was conducted for 48 and 72 h post-treatment, respectively. Subsequently, MTS solution was added to each well and incubated for 4 hours at 37 °C with 5 % CO₂ followed by an absorbance reading at the 490 nm wavelength using Infinite 200 Pro-multiplate reader (Tecan, Männedorf, Switzerland).

Total phenolic content (TPC) was assayed according to the Folin–Ciocalteau method [24 ], with a little modification, using gallic acid as standard. A volume of 1580 μL of distilled water and 100 μL of Folin–Ciocalteu reagent were added to 20 μL of sample extract (or standard solution) and incubated at room temperature for 8 min. Then, 300 μL of sodium carbonate solution (20% w/v) was added. The solution mixture was incubated in the dark at room temperature for two hours. The absorbance at 765 nm was measured in a 96-well plate (Greiner Bio-one, Frickenhausen, Germany) using a multifunctional microplate reader (Tecan Infinite 200 PRO multiplate reader, Männedorf, Switzerland). The estimation of TPC in the samples was calculated by a calibration curve obtained with gallic acid. The results are expressed as mg of gallic acid equivalents (GAE) per g of dry weight (DW) sample.

The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega, WI, USA) was performed to evaluate the cytotoxicity of tested compounds against Vero cells and BHK21 cells according to the manufacturer’s protocol. Briefly, monolayers of Vero cells were grown in 96-well plate and were treated with different concentrations of each compound in triplicate together with negative control (media containing 0.1% DMSO). The plate was then incubated at 37 °C with 5% CO₂ for 48 hours before the MTS assay was performed. Treated and control cells were kept for two days at 37 °C, under similar conditions and duration until used for antiviral activity assay. After two days post-treatment, MTS solution was added to the cells and incubated for 4 hours at 37 °C with 5% CO₂ prior to absorbance detection at 495 nm wavelength using Infinite 200 Pro multiplate reader (Tecan, Männedorf, Switzerland). All experiments were conducted in triplicate. The half maximal cytotoxic concentration (CC₅₀) for each compound was determined through this assay using Graph Pad Prism 5 (Graph Pad Software Inc., San Diego, CA, USA, 2005).