Accuskan fc

AlamarBlue (AB) assays were used to elucidate the inhibitory effect on Cytopathic effect (CPE) induction caused by SARS-CoV-2 infection. Briefly, Vero E6 cells (infected and non-infected) were incubated with Dulbecco's Modified Eagle Medium (DMEM) without FBS or supplements, and AB solution (5% [v/v] solution of AB dye). Following 3 h incubation, AB absorbance was quantified at 600 nm using a Fisher AccuSkan FC microplate reader. Twelve technical replicates per experiment at concentration of 25 nM was carried out (n = 5) for cytopathic effect evaluation of SARS-CoV-2 in the presence of free-hACE2 protein or Lipo-hACE2. The reading was performed in triplicate (the median values obtained from 3 different wells was calculated). The relative cell viability rate (%) was calculated by comparing infected and non-infected wells [(mean fluorescence of treated-SARS-CoV-2 infected wells/mean fluorescence of treated non-infected wells) × 100]. Each assay was carried out in triplicate. To score SARS-CoV-2 induced CPE, we performed microscopy to image the infected cells in the presence or absence of Lipo-hACE2 or free hACE2 treatment.

The bacterial strain used in this study is HB101 a derivative of E. coli K12. It was grown in Luria Broth (LB) containing 10 g/l of tryptone, 5 g/l of yeast extract and 10 g/l of NaCl. The culture media was supplemented with 1 to 20 μCi/ml of [³²P] Na₂HPO₄ (Perkin Elmer, NEX011001MC), 0.1 μM to 1 mM of IPTG and 5 μg/ml of ampicillin depending on the experiment performed. Cultures were initiated from starters grown overnight and diluted 100-fold into either 500 μl in 1.5 ml micro-centrifuge tubes (after poking the cap with a large diameter needle to allow proper aeration) or 250 μl in 96-well micro-plates. Cells were grown aerobically and under strong agitation in an Eppendorf Thermomixer or a shaker-incubator micro-plate reader (Accuskan FC, Fisher Scientific) to midlog phase (OD_600nm = 0.5) prior to sample collection by centrifugation. Competent cells were prepared by washing bacteria twice in ice cold 50 mM CaCl₂, pH 6.1.