Bz x700 all in one fluorescent microscope

Pseudovirus neutralization
was performed as previously described.^{46 (link)} Briefly, 293T-ACE2 cells were seeded at 10 000 cells per
well on tissue culture treated, poly lysine treated 96-well plates.
Cells were grown overnight at 37 °C. LzGreen SARS-COV-2 S pseudotyped
lentivirus was combined with 2-fold serial dilutions of CBDA and CBGA
in DMSO or vehicle control. The virus–drug mixture was incubated
at 37 °C for 1 h, after which virus was added to 293T-ACE2 treated
with 5 μg/mL Polybrene. Cells were incubated with neutralized
virus for 44 h, then fixed with 4% formaldehyde for 1 h at room temperature,
incubated with DAPI for 10 min at room temperature, and imaged with
a BZ-X700 all-in-one fluorescent microscope (Keyence, Itasca, IL,
USA). Total areas of DAPI and GFP fluorescent signal were calculated
using included microscope software (Keyence). To account for variability
in cell count, green fluorescent signal was normalized to DAPI signal.
For conditions with fewer DAPI foci, the modal value of DAPI signal
for each set of replicates was used for normalization across that
condition. Otherwise DMSO control values were used in normalization
to manage DAPI inconsistency across replicates. IC₅₀ values
were calculated with combined replicate data in python using a three-parameter
logistic model and plotted with the matplotlib data visualization
library.

293T cells were seeded at 2 million cells/dish in 6cm TC-treated dishes. The following day, cells were transfected as described above with lentivirus packaging plasmids, SARS-CoV-2 S plasmid, and lzGreen reporter plasmid (Crawford et al., 2020 (link)). After transfection, cells were incubated at 37C for 60 hours. Viral media was harvested, filtered with 0.45μm filter, then frozen before use. Virus transduction capability was then tittered on 293T-Ace2 cells treated with 50μl of 5μg/ml polybrene (Sigma-Aldritch LLC). LzGreen titer was determined by fluorescence using BZ-X700 all-in-one fluorescent microscope (Keyence), a 1:16 dilution was decided as optimal for following neutralization assays due to broad transduced foci distribution.

The neutralization protocol was based on previously reported neutralization methods utilizing SARS-CoV-2 S pseudotyped lentivirus (Crawford et al., 2020 ). 293T-ACE2 cells were seeded poly-lysine treated 96-well plates at a density of 10,000 cells per well. Cells were allowed to grow overnight at 37°C. LzGreen SARS-COV-2 S pseudotyped lentiviruses were mixed with saRBD-1, or VHH52 control antibody. Immunized alpaca serum was used as positive neutralization control, while virus alone was used as negative control. Dilutions of antibodies ranged from 177 nM to 170 pm for saRBD-1 and 26.3nM and 25 pM for Fc-saRBD-1, and 6.57 nM to 4 pM Bi-saRBD-1. Virus-antibody mixture was incubated at 37°C for 1 hour after which polybrene was added up to 5 μg/ml and the mixture was added to 293T-ACE2 cells. Cells were incubated with neutralized virus for 44 hours before imaging. Cells were fixed with 4% PFA for 1 hour at RT. Fixed cells were washed with PBS 2x, then incubated with 10 μg/mL DAPI for 10 minutes at RT, imaged with BZ-X700 all-in-one fluorescent microscope (Keyence). Estimated area of DAPI and GFP fluorescent pixels were calculated with built in BZ-X software (Keyence).

293T cells were seeded at 2 million cells/dish in 6cm TC-treated dishes. The following day, cells were transfected as described above with lentivirus packaging plasmids, SARS-CoV-2 S plasmid, and LzGreen as described above (Crawford et al., 2020 ). After transfection, cells were incubated at 37°C for 48 hours. Viral media was harvested, filtered with 0.45μm filter, then frozen before use. Virus transduction capability was titered on 293T-ACE2 cells in DMEM plus 5 μg/mL polybrene. LzGreen titers were determined by fluorescence using BZ-X700 all-in-one fluorescent microscope (Keyence): a 1:16 dilution was decided as optimal for following neutralization assays due to broad transduced foci distribution. Each well was captured by Keyence microscope and stitched using built-in software. GFP signal was quantified for the stitched images using Keyence software, transduction levels were normalized to virus only control wells.