Luciferase assay system reagent

Custom made analysis plates were fabricated from black 1/8" thick polymethyl methacrylate (PMMA), 3M^TM adhesive transfer tape (double sided, #9770) and thin polycarbonate sheet, all purchased from Piedmont Plastics, Inc. (Beltsville, MD, USA). Luciferase Assay System reagent was purchased from Promega (Sunnyvale, CA, USA). Bacillus cereus toxin was a gift from Toxin Technology (Sarasota, FL, USA). HEK293 cells (ATCC CRL-1573), a cell line originating from human embryonic kidney, and epithelial Vero cells (ATCC CCL-81), a cell line originating from the kidney of African green monkey, were obtained from the American Type Culture Collection (Manassas, VA, USA).

Pseudotyped SARS-CoV-2 particles were provided by the Academy of Military Medical Sciences. For pseudovirus transduction, pEGFP-N1-bACE2-Rm and pEGFP-N1-hACE2 plasmids fused to eGFP were transferred to BHK21 cells. After 24 h, eGFP⁺ cells were sorted by flow cytometry. Then, eGFP⁺ cells (4 × 10⁴ cells per well) were seeded in 96-well plates for 24 h. The TCID₅₀ of pseudovirus particles was serially diluted three folds ranging from 2.8 × 10⁴ to 3.1 × 10⁴. Then, the supernatant containing pseudovirus particles was added to the BHK21 cells after PBS washing. Cells were lysed in the Luciferase Assay System reagent (Promega) at 24 h postinfection. Luciferase activity was detected using a GloMax 96 Microplate luminometer (Promega).

SARS-CoV-2 S-pseudotyped lentiviruses were generated as described in Section 2.1 using pLJM1-Luc2-BSD, pSPAX.2, and pcDNA3-Spike. pLJM1-Luc2-BSD was generated by subcloning Luc2 from the NheI and MfeI sites of pGL4.26 (Promega) into the NheI and EcoRI sites of pLJM1-BSD. pcDNA3-Spike encodes a human codon-optimized S matching bases 21,563 to 25384 from GenBank: NC_045512 (a kind gift from Dr. David Kelvin, Dalhousie University), with a D614G mutation, generated by PCR mutagenesis with primers: 5′-CTGTACCAGGgTGTGAACTGC and 5′-CACGGCCACCTGATTGCT using KOD Xtreme Hot Start DNA Polymerase (Sigma, 71975-M). Pseudotyped lentiviruses were treated with LED light at 49 J/cm² after the addition of 4 µg/mL PhytoQuin or DMSO as described in Section 2.3, then added to HEK293A-ACE2/TMPRSS2 cells and incubated for 24 h at 37 °C. Cells were harvested in 1X Reporter Lysis Buffer (E397A, Promega, Madison, WI, USA) and lysed by freezing at −80 °C. Thawed lysates were added to an Falcon^® white/opaque 96-well plates (25382-208, VWR), mixed with Luciferase Assay System reagent (E1501, Promega) using automated injectors, and read on a FLUOstar Omega microplate reader (BMG Labtech).

Transduction experiments with pseudoparticles were performed on Caco-2 cells. In brief, 10,000 Caco-2 cells were seeded onto the 96-well-flat-bottom plates, one day prior. Peptides were incubated with pseudoparticles at the indicated concentrations for 1 h at 37 °C, before adding the mixtures to cells at 1:10 dilution. Sixteen hours post-transduction, the medium was removed and the cells were washed once with PBS, before adding the Luciferase Cell Culture Lysis Reagent (Promega #E1531, Madison, WI, USA). The lysates were then transferred to white 96-well plates, 50 μL Luciferase Assay System Reagent (Promega #E1500, Madison, WI, USA) was added and substrate conversion was measured as relative light units per second, using the Orion II Microplate Luminometer. The values were corrected for the background signal derived from the untransduced cells.

Luciferase reporter assays were performed 24 h after transfection as previously described [28 (link)]. In more detail, luciferase activity and RFP fluorescence values were determined to utilize a Cytation 5 Plate reader (BioTek, Winooski, VT, USA). Thereby, 100 μL of luciferase Assay System Reagent (Promega, Madison, WI, USA) were injected automatically into each well of a 96 well plate containing 20 μL of cell lysate, the provided cell culture lysing reagent was used as background control. Luminescence was measured for 10 s after a delay of 2 s. Fluorescence was detected with a 544/616 nm filter set.