Glomax discover luminometer

Preparation of SARS-CoV-2 Wuhan-Hu-1 and Omicron BA.1 spike protein-pseudotyped VSVΔG-luc has been described previously (Shinnakasu et al., 2021 (link); Tani et al., 2010 (link); Yoshida et al., 2021 (link)). Briefly, HEK293T cells were transfected with expression plasmids for Wuhan-Hu-1 and Omicron BA.1 spike protein using TransIT-LT1 Transfection Reagent (Mirus) according to the manufacturer’s instructions. After 24 h, cells were infected with VSVΔG-luc virus for 2 h and then washed with DMEM and further incubated for 24 h. Cell-free supernatant was harvested and used for the neutralization assay as described previously (Nie et al., 2020 (link)). Human plasma samples were inactivated at 56°C for 30 min prior to the neutralization assay. Spike-pseudotyped VSVΔG-luc was incubated with serial dilutions of human plasma or recombinant antibodies for 1 h at 4°C, and then inoculated onto a monolayer culture of VeroE6-TMRPSS2 cells (JCRB1819; NIBION) in a 96-well plate. After 24 h, luciferase activity was measured by Luciferase Assay System (Promega) and GloMax Discover luminometer (Promega). The NT₅₀ values for plasma or the half-maximal inhibitory concentration (IC₅₀) values for monoclonal antibodies were determined as described (Nie et al., 2020 (link)).

All 96-well, 384-well and 384-well low volume assay plates were read using the GloMax Discover Luminometer from Promega. The instrument was set to 0.5 s integration time. Data was analyzed using Microsoft Excel and GraphPad Prism, version 8 software. IC₅₀ values were determined by using a nonlinear regression fit to a sigmoidal dose–response (variable slope).
To analyze data from the patient-derived samples and identify samples positive for NAbs, relative luminescence units (RLUs) were converted to net luminescence values by subtracting the “Lumit antibodies-only” control RLU values from luminescence values generated by the negative pre-pandemic and patient-derived COVID-19 PCR positive samples. Then, the net luminescence values of all samples were converted to percent PPI activity using the “Lumit antibodies-only” and the average “Pre-COVID-19 negative serum” controls as 0 and 100%, respectively. The % neutralization or inhibition is calculated using the following formula $$\text{Sample}\%\text{Neutralization}=100-\%\text{PPI activity}.$$ By testing multiple PCR negative samples, we established a cutoff of 30% neutralization that should be sufficient to indicate if a sample is positive for NAbs.

RenG expression strains were grown for 72 h on sBHI agar plates supplemented with kanamycin. Bacteria were suspended in liquid sBHI medium with a range of theophylline concentrations (0 mM, 0.0078 mM, 0.0156 mM, 0.03125 mM, 0.0625 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM) at an OD₆₀₀ of 0.1 and then incubated in an anaerobic chamber at 37°C for 20 h. Coelenterazine-h solution (Prolume, Pinetop, AZ) was added to each sample (3.4 μg mL⁻¹), and luciferase activity was measured on a Promega Glomax Discover luminometer. Optical densities were measured immediately after measuring luciferase activity for normalization. Normalized activity was expressed as relative light units (RLU), which is the luminescence value/OD₆₀₀.

CREB activity was determined by quantifying the luciferase reporter activity in cells transfected with CREB-LUC (α-168), which contains 168 bp of the endogenous alpha polypeptide glycoprotein hormones gene (CGA) promoter containing two identical repeats of the 8-bp core CRE^{52 (link),53 (link)}. Plasmid transfection was performed by electroporation using an Amaxa Nucleofector-1.5. 1 × 10⁶ VSMCs were transfected with 5 μg of DNA or 100 pmoles of siRNA using the standard Nucleofector program A033. Cells were stimulated with the indicated agents 24 hours post transfection followed by lysis in Promega cell culture lysis buffer or for secreted nano-luciferase reporters cell culture media collected and assays using NanoGlow assay system (Promega). Luciferase and Renilla activity were quantified using the dual reporter assay kit (Promega) according to the manufactures instructions using Glomax Discover luminometer (Promega).

5′-UTRs with or without the alternative exons of Hnrnph2, Anapc10, Pex2 and Cwc22 were cloned into psiCheck1 (kindly provided by Dr Aaron Goldstrohm at the University of Minnesota Twin Cities; (22 (link))). These recombinant luciferase reporters were transfected into Tsc1^−/− MEFs using Lipofectamine 3000 (Thermo Fisher Scientific). Eighteen hours after transfection, the luciferase activity was measured using Dual-Glo reagent with the Glomax Discover luminometer (Promega). Expression of Renilla luciferase mRNA was measured to normalize the luciferase activity by RT-qPCR using the following primers: forward 5′-TCTCGTTAAGGGAGGCAAGC-3′ reverse 5′-TGGAAAAGAACCCAGGGTCG-3′. Four replicates of the measurement were conducted for technical repeats.