Glomax explorer system

Apoptosis and cell death was analysed via FACS using the Guava® easyCyte™ Flow Cytometer (Luminex, US), as described elsewhere [67 (link)]. For analysis of caspase 3/7 activity, cells were seeded at a density of 1 × 10⁴ cells/well, cultured for 24 h prior to addition of 100 μl of Caspase-Glo® 3/7 reagent directly to cells, incubation for 1 h at RT and recording of luminescence using the GloMax® Explorer system (Promega, UK).

Cell lines HEK293T, IL16lamp2b and EXO-Tat were cultured in Dulbecco’s Modified Eagle medium (GE Healthcare Bio-Sciences, PA, USA) with 10,000 U/mL penicillin-streptomycin (Thermo-Fisher, USA), 200 mM of L-Glutamine (Thermo-Fisher, USA), and fetal bovine serum over a period of 48 hours. Each of the cell lines were cultured in five plates and considered as five replicates. After dissociation with 0.25% trypsin (Thermo-Fisher, USA), cells were centrifuged and re-suspended in PBS buffer (GE Healthcare Bio-Sciences, PA, USA). An equal number of cell (3.0 × 105/mL) concentrations were used for each assay.
Cell Viability assay was preformed using the CellTiter-Glo 2.0^® bioluminescence assay kit (Promega, WI, USA) according to the manufacturer’s protocol. Readings were taken using the Glo-Max Explorer system (Promega, WI, USA), wherein higher luminescence correlated to viability [31 (link)]. Readings were taken using the Glo-Max with higher luminescence correlating to more cell death [32 (link)]. Reactive Oxygen Species (ROS) were measured using ROS-Glo™ H₂O₂ assay kit (Promega, WI, USA) according to the manufacturer’s protocol wherein higher signals are indicative of increased amount of ROS in cells [33 (link)].

Viral cytopathic effect (CPE) was examined at 24-h postinfection using the MTS assay as described above. After A/PR/8/34-GFP virus infection, viral GFP expression was analyzed by a Nikon ECLIPSE Ti fluorescence microscope (Nikon, Tokyo, Japan), and GFP intensity was determined using a GloMax Explorer System (Promega, Madison, WI). We calculated 50% effective concentrations (EC₅₀) values for TSN and OCN by regression analysis.

ROS generation was determined with a fluorescent probe, dichlorofluorescein diacetate (DCFH–DA). HepG2 cells were pre-incubated with BSE for 3 h and then incubated with tert-butyl hydroperoxide (t-BHP) for another 1 h. The cells were stained with 10 DCFH–DA (μM) by incubating for further 1 h. The fluorescence intensity in the cells was measured using a Glomax Explorer System (Promega, Madison, WI, USA). ROS production was calculated relative to the vehicle-treated control.

Two μg of NotI‐linearized plasmid DNA was transcribed in vitro using T7 RNA polymerase (Thermo Fisher Scientific) according to the manufacturer's protocol. RNA was purified using a GeneJET RNA purification kit (Thermo Fisher Scientific). The concentration and quality of the RNA were measured using BioPhotometer (Eppendorf). Four μg of RNA was subjected to in vitro translation using the Rabbit Reticulocyte Lysate System (Promega Corporation) according to the manufacturer's instructions. Luciferase activity was then measured using the Luciferase Assay System (Promega Corporation) in the GloMax Explorer System (Promega Corporation).

HEK293 cells were transfected with 500 ng/well of plasmid constructs expressing luciferase using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol, at around 75–90% confluency in a 24‐well plate. A plasmid expressing Renilla luciferase was co‐transfected (100 ng/well), serving as the transfection control. Cells were lysed 24 h post‐transfection, and luciferase activity was measured using Dual‐Luciferase Reporter Assay System (Promega Corporation) in the GloMax Explorer System (Promega Corporation). For the readthrough assay in the presence of miRNA inhibitors, HeLa cells were transfected with 10 nM of let‐7a inhibitor or control inhibitor (Sigma) along with the luciferase constructs. The cells were lysed 48 h post‐transfection, and the luciferase activity was measured as described above. In all the constructs (including mutations and deletions), FLuc coding sequence was in‐frame with AGO1 (or RHOA) coding sequence.