Biotek lionheart fx automated microscope

Pseudovirus from SARS-CoV-2 B.1.617.2 (Cat. No. FNV3718) and B.1.1529 (Cat. No. FNV4122) were purchased from FUBIO (Jiangsu, China). Pseudovirus (2×107 TFU/ml) were pre-incubated with different concentration of OA or ACE2-Fc (GenScript, Cat. No. Z03516, Nanjing, China) as a positive control in a 96-well plate for 1 h at room temperature. ACE2^h cells were then seeded at 3×10⁴/well into the plate with the pseudovirus-OA mixture and incubated for additional 6 h. Then the medium was replaced with fresh medium and incubated for another 48 h. The GFP images were captured by BioTek Lionheart FX Automated Microscope (Agilent Technologies, USA). Cells were harvested and the luciferase activity was measured using the luciferase reagent (Promega, Shanghai, China, Cat. No. G7941) by the microtiter plate reader (PerkinElmer, Victor XLight). The inhibition rate was calculated as the following formula: Inhibition rate = (1- Signal of positive control—Blank control/Signal of Negative control—Blank control) × 100%.

The plates used were 96-well plates with a U-shaped bottom and a less adherent surface. Cells were seeded onto a plate at a concentration of 3000 cells/well. The plates were centrifuged at 2000 rpm for 5 min, and then the prepared cells were left for 3 days in an incubator (37 °C, 5% CO₂) to form a 3D structure [9 (link)]. On the fourth day, treatment was performed. After 48 h, 5 μL of calcein and 1 μL of propidium iodide (IP) from a Cellstain double staining kit (Sigma Aldrich, Darmstadt, Germany) were added to each well. Fluorescence intensity was measured using the Agilent BioTek Lionheart FX Automated Microscope (Agilent Technologies, Santa Clara, CA, USA) at excitation (about 501 nm) and emission (about 521 nm) for calcein and at excitation (about 488 nm) and emission (about 617 nm) for IP.