Glomax multi microplate luminometer

Eukaryotic expression vector pCIwt-NS1 encoding wt-NS1 was constructed by sub-cloning its coding sequence between the XhoI and NotI sites of the pCI plasmid (Promega, Madison, WI, USA). In order to prevent production of spliced mRNAs, splice-donor and splice-acceptor sites were both invalidated by point mutations [33 (link)]. Substitutions within NS1 (R38A-K41A) were introduced using the QuikChange II site-directed mutagenesis kit. Subconfluent HEK293T cells in 24-well plates (one technical triplicate for each condition) were transfected using the FuGENE (Promega, Madison, WI, USA) reagent with the expression vectors of the four viral polymerase subunits (PB1, PB2, PA, and NP) and the pPolI-NS-Renilla encoding the chimeric minigenome (consisting of the NS genome segment with NS1′s Open Reading Frame (ORF) replaced by the Renilla Luciferase ORF), along with the pCI-NS expression vector (or empty vector as a control). The pCMV-Firefly plasmid (Promega) was used as control for transfection efficiency. Twenty-four hours post-transfection, cells were lysed and the activities of the two luciferases were measured using the Dual-luciferase reporter assay system (Promega) and a GloMax-Multi microplate luminometer (Promega). The minireplicon-driven Renilla-luciferase activity was normalized with respect to the activity of the Firefly luciferase, which was used as a transfection control.

The cell viability was tested by CCK-8 assay (ab228554; Abcam) according to the manufacturer’s instruction. WST-8/CCK8 tetrazolium salt is reduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living and metabolically active cells and is measured by absorbance at 460 nm. Therefore, Vero cells (2.5 × 10⁴ cells/mL) were grown in 96-well microtiter plates at 37 °C in a 5% CO₂ incubator for 24 h. Then, they were exposed to serial dilutions of BSE (50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL, and 300 μg/mL) for 72 h and incubated with CCK8 tetrazolium salt for 4 h at 37 °C in a CO₂ incubator. The absorbance was measured at 460 nm with GloMax^® Multi Microplate Luminometer (Promega Corporation, 2800 Woods Hollow Road, Madison, WI, USA) and the % of cellular viability was calculated compared to untreated cells.

The cell viability of HEp-2 cells treated with DOX, GCD and GCD@DOX was determined on the basis of ATP levels using ViaLightTM plus cell proliferation and cytotoxicity bioassay kit according to the manufacturer’s instructions (Lonza Group Ltd., Basel, Switzerland). Cells were grown in 96-well plates and treated with different concentrations of indicated DOX (1.25 μg/mL), GCD (25 μg/mL) and GCD@DOX (25 μg/mL). The contents of DOX in 25 μg/mL of GCD@DOX was 0.625 μg/mL. After the indicated incubation time, the cells were harvested and the emitted light intensity related to ATP degradation was quantified with the GloMax Multi Microplate Luminometer (Promega Corporation, 2800 Woods Hollow Road Madison, WI, USA). The luminescence value was converted to the cell proliferation index and reported as percentage of cell viability (%) according to the following equation:
where A denotes the average of treated sample, B represents background luminescence, and C represents the average of untreated samples.