Spectramax m5

Pseudovirus of prototype strains, and BA.1, BA.1.1, BA.2, BA2.12.1, BA2.75, BA.4/5, BF.7, BQ.1, and XBB were generated by co-transfection of 293 T cells with pLenti-Luciferase, psPAX2 and viral S protein expression plasmids, pCAGGS-prototype-S, pCAGGS-BA.1-S, pCAGGS-BA.1.1-S, pCAGGS-BA.2-S, pCAGGS-BA.2.12.1-S, pCAGGS-BA.2.75-S, pCAGGS-BA.4/5-S, pCAGGS-BF.7-S, pCAGGS-BQ.1-S, and pCAGGS-XBB-S. Neutralization assays were performed as follows. Briefly, 293T-ACE2 cells were seeded in a 96-well plate at a concentration of 1 × 10⁴ cells per well in 100 μL of DMEM with 10% FBS and cultured for 12–16 h. Fivefold serially diluted plasma starting at 1/40 from recovered patients were incubated with SARS-CoV-2 pseudotyped virus for 1 h at 37 °C. The mixture was added to 293T-ACE2 cells. After 48 h post-infection, luciferase activity was measured with a luciferase assay system (Promega, E1501) on a multifunctional microplate reader SpectraMax M5. The titers of SARS-CoV-2–specific NAb were calculated as a 50% half-maximal inhibitory does (ID50) and expressed as the dilution of plasma that resulted in a 50% reduction of luciferase luminescence compared with virus control in single-round pseudovirus infection assay. The cut-off for a positive NAb titer was 1/80.

Pseudovirus of prototype strains, and BA.1, BA.1.1, BA.2, BA2.12.1, BA2.75, BA.4/5, BF.7, BQ.1, and XBB were generated by co-transfection of 293 T cells with pLenti-Luciferase, psPAX2 and viral S protein expression plasmids, pCAGGS-prototype-S, pCAGGS-BA.1-S, pCAGGS-BA.1.1-S, pCAGGS-BA.2-S, pCAGGS-BA.2.12.1-S, pCAGGS-BA.2.75-S, pCAGGS-BA.4/5-S, pCAGGS-BF.7-S, pCAGGS-BQ.1-S, and pCAGGS-XBB-S. Neutralization assays were performed as follows. Briefly, 293T-ACE2 cells were seeded in a 96-well plate at a concentration of 1 × 10⁴ cells per well in 100 μL of DMEM with 10% FBS and cultured for 12–16 h. Fivefold serially diluted plasma starting at 1/40 from recovered patients were incubated with SARS-CoV-2 pseudotyped virus for 1 h at 37 °C. The mixture was added to 293T-ACE2 cells. After 48 h post-infection, luciferase activity was measured with a luciferase assay system (Promega, E1501) on a multifunctional microplate reader SpectraMax M5. The titers of SARS-CoV-2–specific NAb were calculated as a 50% half-maximal inhibitory does (ID50) and expressed as the dilution of plasma that resulted in a 50% reduction of luciferase luminescence compared with virus control in single-round pseudovirus infection assay. The cut-off for a positive NAb titer was 1/80.

After treatment, cells were transferred to 96-well plates (20,000 cells/well) with 60 μL fresh-media and 20 µL of H₂O₂ substrate solution from ROS-Glo^TM H₂O₂ assay kit (Promega) and were cultured for 4 h. ROS-Glo™ detection solution was added to cells and incubated for 20 min, and Lum was recorded for 0.5 sec integration using the SpectraMaxM5 spectrophotometer.

Cells were seeded at 80,000 cells/cm² density and transfected with 100 ng/well of pGL4.13 luciferase plasmid using either Lipofectamine 3000 or Viromer RED as described above. Nontransfected cells served as a negative control. At 24 hr after transfection, following viability estimation using the CellTiter-Fluor kit (Promega) as described above, the media was aspirated, the wells were washed with DPBS (pH 7.4) and 1X passive lysis buffer (Promega) was directly added to the wells for incubation at room temperature for 15 minutes to lyse the cells. Typically, 20 μL of 1X passive lysis buffer (reconstituted in water) was added to each well of the 96-well plate and subjected to brief agitation on an orbital shaker. After cell lysis, 100 uL of luciferase assay reagent (Promega) was added to each well and the measurement of luminescence was performed using a microplate reader (SpectraMax M5) at 37°C with 1,500 milliseconds of integration time. Readings were taken at least twice and averaged. Average luminescence (in relative light units; RLU) was divided by the mean RFU obtained for the same well from using the CellTiter-Fluor kit (Promega). Resultant values were further normalized to the mean RLU/RFU ratio of the nontransfected control for each category and reported as a fold change.

The luciferase-encoding HIF1α-promoter or VHL-promoter and control sequences were purchased from Hanbio Biotechnology (Shanghai, China) Co., Ltd. Cells were plated in a 96-well plate, transfected with luciferase, and at 48 h after transfection, the culture medium was renewed. Luciferase activities were measured by a commercial kit (SpectraMax M5) using the Dual-Luciferase Reporter Assay System (Promega, USA).

HCT116 cells were seeded at the density of 2 × 10⁵/well in 48-well plates, NFATc2-luciferase reporter was cotransfected with pCDNA3.1-p53 plasmid using Lipofectamine 2000. Cells were incubated at 37 °C with 5% CO₂ for 24 hours. After replacement with fresh medium, cells were treated without or with 10 μM As₄S₄ for 12 hours. Luciferase activities were determined using a Dual-Glo Luciferase Assay System (Promega) in the SpectraMax M5 (USA).