Fluoroshield mounting media

HepG2-hNTCP-C4 cells (~5 × 10⁴ cells) were grown on 12-mm coverslips coated with 0.1 mg/mL collagen (Sigma) for 24 hours, washed with 1 × PBS and incubated with either 1 μM preS1(60)-FITC protein or with preS1(60)-FITC -scFv mixtures at room temperature for 30 min. The preS1(60)-FITC protein (1 μM) and the scFv (10 μM for each scFv or control antibodies) were pre-equilibrated for 1 hour at room temperature in DMEM containing 0.5% FBS. (For Cocktail, each scFv was taken at a concentration of 2.5 μM to get a final cocktail concentration of 10 μM). The cells were washed with PBS three times, fixed with 4% (w/v) paraformaldehyde (PFA) and the coverslip was mounted using Fluoroshield^™ mounting media with DAPI (Sigma, F6057) on a glass slide. The fluorescence microscopy was performed on an Olympus Laser Confocal Scanning Microscope (FV1000D) using a 60 × oil objective.
For flow cytometry experiments, 5 × 10⁴ cells were seeded in collagen coated 24-well plate (Corning, 354408), grown at 37 °C for 24 h, treated and fixed similar to confocal microscopy. The samples were acquired using FACS Verse flow cytometer (BD Biosciences) and the analysis was performed on FlowJo software.

Samples that were subjected to tensile testing were imaged by two-photon confocal microscopy, using a custom Prairie View Ultima multiphoton microscope (Bruker Corp., Billerica, MA, USA) to localize CF-CHP binding within the tissue. Fascicles were mounted in Fluoroshield mounting media (Sigma-Aldrich, St Louis, MO, USA) and imaged with 910 nm excitation and detection in 435–485 nm and 500–550 nm for collagen SHG and CF two-photon excited fluorescence, respectively.

HEK293T-ACE2 cells were seeded at a density 9 x 10⁴ cells/well of an 8-well μ-Slide (Ibidi, 80826) in 250 μL complete media. After 1 h, cells were transfected in duplicate with the indicated FlipGFP-based reporter constructs as above. The following morning, cells were infected with SARS-CoV-2 at the indicated MOI and incubated for 24 h.
To measure reporter activation in infected cells using confocal microscopy, cells were first fixed for 15 min by incubation in 4% PFA. Cells were then permeabilised with Perm/Wash buffer (BD), stained for SARS-CoV-2 spike protein using a mouse monoclonal antibody (GeneTex, GTX632604) for 30 min at room temperature, washed twice, stained with an anti-mouse AF647 secondary antibody (Jackson ImmunoResearch, #715-605-150) for 30 min at room temperature, mounted with 200 μL/well of Fluoroshield Mounting Media (Sigma, F6057), and analysed by confocal microscopy using a Zeiss LSM 710 Inverted confocal microscope equipped with 405, 458, 543 and 633 nm lasers and a Plan Apochromat 63X/1.40 Oil DIC M27 objective. For each reporter construct, the ratio of FlipGFP/mCherry MFI was calculated manually using Fiji (ImageJ) [43 (link)], by creating a mask around syncytiated cells that were both spike+ (infected) and mCherry+ (transfected).