Luc pair firefly luciferase hs assay kit

The promoter fragments of IGFBP3 (+ 160/ − 1414, + 160, 5′-AAACTCGAGGCATTCGTGTGTACCTCGTG-3′;–1414, 5′-CCCGAGCTCTGATCTTCCCCTGTCCACTC-3′), HIF-1a (+ 80/ − 1871, + 80, 5′- AAACTCGAGCTCTCCTCAGGTGGCTTGTC-3′;–1871, 5′- CCCGAGCTCGAGTTGCAGTGAGCCGAAAT-3′), and HIF-2a (+ 46/-1222, + 46, 5′-AAACTCGAGGAGGACAAGCTGGCAGAGAC-3′;–1222, 5′- CCCGAGCTCGTGTTCCGCATTTTGGAAGT-3′) were generated by chromosomal DNA extraction from P0, amplified by Q5 High-Fidelity DNA Polymerase ((NEB, Ipswich, MA, USA), and constructed into pGL3 (Promega, Madison, WI, USA). The pGL3-IGFBP3, pGL3-HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 10⁴ cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA). Luciferase activities were calculated relative to the empty pGL3 transfectants.

GMRαβ or CKO HeLa cells were seeded into 24-well plates for transfection. Reporter plasmids pGL4-CISH or pCISHmut (50 ng) were co-transfected with the Renilla luciferase reporter plasmid (0.5 ng) and/or other indicated plasmids. After transfection, cells were maintained in DMEM culture medium for 24 h, and then changed to fresh serum-free DMEM with or without 10 nM GM-CSF for an additional 24 h. Cells were then harvested and assayed for luciferase activity using Luc-Pair™ firefly luciferase HS assay kit (GeneCopeia, Germantown, MD, USA). Briefly, the cells were washed in PBS and lysed with Luc-Lysis II Buffer at room temperature for 15 min, and then analysed for firefly luciferase activity (FLuc assay working solution) and Renilla luciferase activity (RLuc assay working solution). Each assay was performed at least four times.

Neutralization assays were performed in TZM-bl cells, as described previously [51 (link)]. To use TZM-bl cells, antibiotics were removed from virus stocks by growing them in GHOST cells in the absence of antibiotics. This process also removed antibodies present in the culture supernatants. Virus-containing supernatants were harvested at 48–72 hpi, spun to remove debris, aliquoted, and used for neutralization assays. Viruses were incubated with 0–10 ug/ml of antibodies at room temperature for 30 minutes and then added to TZM-bl cells plated at 10,000 cells per 96 well one day before the assay. At 48 hpi, infection levels were measured by luciferase assays using Luc-Pair Firefly Luciferase HS assay kit (GeneCopoeia). The relative light unit was read at 575 nm using a Victor X3 plate reader (PerkinElmer).

A PV entry (transduction) assay was performed by incubating Mock-293T or hACE2/hTMPRSS2-293T cells on 48-well plates with PVs (5 × 10⁸ genome copies in 200 μL per well) for 1 h at 37°C in a CO₂ incubator. Medium was replaced with DMEM containing 10% FBS. The entry levels of PVs expressing firefly luciferase were assessed by measuring luciferase activity using the Luc-Pair firefly luciferase HS assay kit (catalog number LF009; GeneCopoeia) and reading the plates on the SpectraMax paradigm multimode detection platform using SoftMax Pro 6.3 (Molecular Devices).

On day 14 and 35 post primary immunization, mice anesthetized with isoflurane were bled via the retro-orbital route in heparinized micro-hematocrit capillary tubes. Plasmas were collected by spinning the blood at 1500 g for 15 min at 4 °C. Individual plasma (n = 10 per group) was heat-inactivated at 56 °C for 30 min and serially diluted in 50 μl RPMI media containing 1 μg/ml puromycin and 10% FBS. Then 5 × 10^7 genome copies of PV expressing Fluc in 50 μl RPMI 1640 media were preincubated with the serially diluted plasmas at RT for 30 min. Preincubated samples were transferred onto ~60% confluent H1299-hACE2 cells on 96-well plates. Neutralizing ability of plasmas were assessed 24 h later by measuring firefly luciferase activity using the Luc-Pair Firefly Luciferase HS Assay Kit (Cat# LF009, GeneCopoeia). Neut₅₀ was calculated through log₁₀ transformation of the plasma dilution factors, at which 50% of neutralization was obtained, using default settings for log(inhibitor) vs. response variable slope method (four-parameter model) in GraphPad Prism version 9.0 (GraphPad Software lnc.).