D8537

Recombinant biotinylated SARS-CoV-2 spike Spike-receptor-binding domain with a
C-terminal human IgG Fc domain fusion (referred to as Spike-RBD) was prepared as
previously described^{50 (link)}. Calu-3 cells
were grown in 96-well flat bottom plates until >50% confluent. Media was aspirated
and cells were washed once with PBS. Cells were then treated with TrypLE to release them
from the plate, RPMI 1640 media was added to dilute TrypLE, and cells were pelleted by
centrifugation at 200×g for five minutes. From this point on, all steps were
carried out on ice. Cells were incubated in 3% BSA (Sigma Aldrich A7030) in DPBS
(Sigma-Aldrich D8537) for 15 minutes to block and washed twice in 3% BSA in DPBS by
centrifugation at 200×g for five minutes in v-bottom plates, followed by
resuspension. Spike-RBD was diluted in 3% BSA to appropriate concentrations and incubated
with cells for 30 minutes on ice. Cells were then washed twice with 3% BSA in DPBS and
incubated with Anti-Strep PE-Cy7 (Thermofisher SA1012) at 5 μg/mL. Cells were
washed twice and subjected to flow cytometry on a FACS Celesta in HTS mode. Cells were
gated to exclude doublets and the median PE-Cy7 signal was calculated for each sample. The
gating strategy is shown in Supplemental
Figure 2. EC₅₀ values and their 95% confidence intervals were
calculated by fitting the RBD binding data into a Sigmoidal, 4PL model in Prism 6.

The primary antibodies were conjugated with fluorescent dyes using a rapid conjugation kit (ab269823, Abcam). For each 10µL of primary antibody, 1µL of modifier reagent was added and mixed gently. The lyophilized powder was dissolved in 10µL PBS (D8537, Sigma-Aldrich), and 1µL of this solution was added to each antibody and mixed gently. The mixture was incubated at room temperature for 15 min in the dark. After incubation, 1µL of quencher reagent was added for each 10µL of antibody used and was then gently mixed. In this experiment, the antibodies were conjugated to Alexa Fluor 647, which is a bright dye with less background fluorescence^{25 (link)}. While Alexa Fluor 488 is the brightest among the dyes, the channel has higher background fluorescence. Alexa Fluor 555 is the weakest dye, suitable for staining proteins with high abundance and high affinity.

Cells were seeded on coverslips at the desired confluence. After 24 h, plates were washed with PBS (D8537, Sigma Aldrich) and incubated in the dark for 30 min with 2 µM BODIPY 493/503 (D3922, Thermo Fisher Scientific) staining solution prepared in ECGM2. Coverslips were washed 3 times in PBS, fixed in 4% PFA for 10 min, stained with DAPI for another 5 min, washed in PBS, and mounted in a drop of Prolong^® Gold (P36934, Thermo Fisher Scientific). Images were acquired on the Nikon C2 Eclipse Ni-E inverted confocal microscope, the super resolution LSM 880 AiryScan, and the Olympus IX73. Analysis was performed using NIH ImageJ software. For corneal tissue staining, dissected corneas were washed in PBS followed immediately by staining with 2 µM BODIPY 493/503 staining solution prepared in PBS, for 30 min in the dark. Corneas were washed for 30 min at room temperature, fixed in 70% ethanol for 1 h, washed with PBS, stained with DAPI for 10 min, and flat-mounted on a microscope slide. Images were acquired on the super resolution LSM 880 AiryScan and the Zeiss LSM780 microscope.