Spectramax id3 plate reader

Plasmid pcDNA3-TEV-FlipGFP-T2A-mCherry was ordered from Addgene (catalog No.124429). pcDNA3 FlipGFP-M^pro plasmid and pcDNA3 FlipGFP-PL^pro plasmid were constructed by introducing SARS-CoV-2 M^pro cleavage site AVLQSGFR and SARS-CoV-2 PL^pro cleavage site LRGGAPTK, respectively, via overlapping PCRs. pLVX SARS-CoV-2 M^pro and pcDNA3.1 SARS-CoV-2 PL^pro plasmids was ordered from Genescript (Piscataway NJ) with codon optimization.
The Flip-GFP M^pro and PL^pro assays were performed as previous reported [15 (link), 23 (link), 24 (link), 37 (link)]. Briefly, the assay started with seeding 293T-ACE2 in 96-well, black, clear bottomed plate (Greiner, catalog No.655090) and incubating overnight to allow cells to reach 70–80% confluency. 50 ng of pLVX SARS-CoV-2 M^pro (or pcDNA3.1 SARS-CoV-2 PL^pro) and 50 ng of pcDNA3 FlipGFP- M^pro (or pcDNA3 FlipGFP-PL^pro) reporter plasmid was mixed with transfection reagent TranslT-293 (Mirus, catalog No. MIR 2700). The mixture was then transfected to each well according to manufacturer’s instructions. After 2.5-3 hours of incubation in 37°C, 1 μL of testing compound was added into each well directly and mixed by gentle plate shaking. 48 hours post transfection, fluorescence was quantified using SpectraMax iD3 plate reader (Molecular Devices) and images were taken using BZ-X800E fluorescence microscope (Keyence) in GFP and mCherry channels at 4X objective lens.

For serum preparation, whole blood, which was collected directly from the heart of sedated mice, was allowed to clot and serum was separated from plasma by centrifugation. Mouse cxcl1/kc DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) was performed to analyze serum kc level according to manufacturer’s instructions. Serum samples were utilized in a 1:10 dilution and final OD was measured at 450 nm against a reference wavelength of 540 nm on a SpectraMax iD3 plate reader (Molecular Devices, San Jose, CA, USA).

After the second application of WEL-DS and before O₃ exposure, a part of tissues (N = 3 per condition) were incubated in the dark with 340 μM 2′,7′-dichlorofluorescein diacetate (DCF-DA) in DPBS for 30 min at 37 °C in a humidified 5% CO₂ atmosphere, to determine intracellular ROS levels. Then, tissues were rinsed with DPBS, placed in a new plate with culture medium and exposed to O₃ 0.4 ppm for 4 h. At the end of O₃ exposure, DCF fluorescence, a measure of ROS production, was determined by SpectraMax iD3 plate reader (Molecular Devices, LLC., San Jose, CA, USA), at 485 nm (excitation filter) and 530 nm (emission filter).

The functional expression of AAV-delivered NLuc gene was tested in vitro using U251 cell line and in vivo using U251 cell line-derived intracranial xenografted NSG mouse model, respectively. For in vitro evaluation, 5 × 10⁴ cells/mL of U251 cells were used to seed 96-well plates and transduced with AAV at different MOIs, i.e. 1,000 to 7,500. Then 25 µM of substate ViviRen (Fisher) was added to the cells expressing AAV-delviered genes three days after transduction. The bioluminescence generated by the expressed NLuc protein was monitored with In Vivo Imaging System (IVIS) Lumina Series III (PerkinElmer, Waltham, MA, USA) [33 (link)]. The 6-well plate was seeded with 5 × 10⁵ cells/mL of U251 cells, transduced with AAV at different MOIs of 0, 1 × 10⁴ and 1 × 10⁶, and induced with 25 µM of substate ViviRen. The dynamic bioluminescence signal was detected with SpectraMax iD3 plate reader (Molecular Devices, San Jose, CA, USA). To detect in vivo expression of AAV-delivered NLuc gene, the U251 xenografted NSG mice received 1 × 10¹⁰ vg of AAV and 37 μg of ViviRen substrate through intracranial injection at the same coordinate of cells xenograft. The NLuc expression (i.e. bioluminescence) was detected in live animals using IVIS Lumina.

Mini-genome assays were performed using human embryonic kidney 293T (HEK 293T) cells cultured in 6-well plates. Cells were transfected with reverse genetics plasmids (1 ug/plasmid mixed with TransIT-LT1 (Mirus)) encoding NP, PA, PB1, and PB2 or PB2 271T, of A/mink (H5N1) to assess the polymerase activity of wild-type PB2 in comparison to the polymerase carrying PB2 A271T. Cells were also transfected with Gaussia Luciferase (GLuc) and Secreted Alkaline Phosphatase (SEAP) reporter plasmids (generously provided by Dr. Daniel Perez, University of Georgia, Athens, GA) and then incubated at 33 or 37 °C^{46 (link)}. Supernatants were collected at 24 h post-transfection, and luciferase and SEAP activity were assayed using a Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia, Inc.) and a SpectraMax iD3 plate reader (Molecular Devices). Polymerase activity was expressed as a ratio of luminescence to SEAP activity for each sample. Three independent experiments were performed in triplicate and results were analyzed using an unpaired, two-tailed Student’s t test.