Biotek synergy neo2

For ATP, overnight cultures were diluted into fresh TSB (OD₆₀₀∼0.05), grown for 4 hr at 37 °C with shaking at 180 rpm (OD₆₀₀∼4), diluted (OD₆₀₀∼1.0) in fresh TSB, and incubated at room temperature with reagent for determination of ATP using BacTiter-Glo Microbial Cell Viability Assay (cat. no. G8232; Promega, Madison, WI), according to the manufacturer’s instructions. Luminescence was detected in a BioTek Synergy Neo2 plate reader (Agilent, Santa Clara, CA). The amount of ATP was calculated and normalized to cell count.
For NAD⁺ and NADH, overnight cultures were diluted into fresh TSB (OD₆₀₀∼0.05), grown for 4 hr at 37 °C with shaking at 180 rpm (OD₆₀₀∼4), and plated for viable counts at 4 hr or concentrated by centrifugation at 12,000 × g for 10 min and resuspended in lysis buffer provided by the assay kit. Cells were lysed by repeated homogenization (two cycles of 45 s homogenization time at 6 M/s followed by a 5 min pause on ice) using Lysing Matrix B tubes in a FastPrep-24 homogenizer (MP Biomedicals, Irvine, CA). After lysis, cell debris was removed by centrifugation (12,000 × g, 10 min), and the supernatant was used for determination of NAD⁺ and NADH levels using a colorimetric assay kit (cat. no. KA1657; Abnova, Taipei City, Taiwan) and a microplate reader (BioTek Synergy Neo2, Agilent, Santa Clara, CA) according to the manufacturer’s instructions.

The bi-molecular fluorescence complementation (BiFC) as been described previously*. Briefly, the HEK293T target cells were transfected using Jetprime with plasmids encoding the GCN4 leucine zipper-Venus1 (ZipV1, kind gift of Stephen W. Michnick, McGill University*), Ace2myc (pCEP4-myc-ACE2 was a gift from Erik Procko (Addgene plasmid # 141185)* and TMPRSS2 (from MISSION TRC3 Human LentiORF Puormycin Library, MilliporeSigma). The HEK293T effector cells were transfected using Jetprime with plasmid encoding GCN4 leucine zipper-Venus2 (ZipV2, also from Stephen W. Michnick) and pCAGGS encoding SARS-CoV-2 S WT, Q957L, or pCAGGS vector. Twenty-four hours post-transfection, cells were washed with PBS and detached with versene (PBS, 0.53 mM EDTA) and resuspended in serum- and phenol red-free DMEM. Wells of a 384-well black plates with optical clear bottom were seeded with effector and target cells (35,000 cells of each populations per well) and incubated for 3 h at 37 °C, 5% CO2. BiFC signal was acquired using Biotek Synergy Neo2 plate reader (BioTek Instruments) using monochromator set to excitation/emission of 500 and 542 nm.

The enzyme activities of GCK in cells were assessed by using commercial assay kits (Abcam, Cambridge, UK) according to the manufacturers’ instructions. GCK converts glucose into glucose-6-phosphate and produces a series of intermediates (NADPH) which could be detected by the probe, generating an intense fluorescence product (Ex/Em = 535/587 nm). Briefly, the cell lysate was diluted by GCK assay buffer (Tris-HCl buffer, pH 8.0). The reaction medium included Tris-HCl, pH 7.4, MgCl₂, dithiothreitol, 0.1% bovine serum albumin, KCl, glucose, nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate dehydrogenase and probe for NADPH. The definition of one unit of glucokinase activity is the amount of enzyme that catalyzes the release of 1.0 µmol of NADPH per minute at pH 8.0 and room temperature. The fluorescence was measured by a microplate reader (Biotek SynergyNeo2, BioTek, Vermont, VT, USA).