2-(beta-(4-hydroxyphenyl)ethylaminomethyl)tetralone

Neutralization assays on the Canadian Blood Services samples used in Figure 2 were performed by 2 independent laboratories, the NML of the Public Health Agency of Canada, and the Wadsworth Center, New York State Department of Health. The cytopathic effect–reduction neutralization assay on the recombinant GenScript antibody was performed in Toronto.
For the PRNT assay at NML, SARS-CoV-2 (Canada/ON_ON-VIDO-01-2/2020, EPI_ISL_42517) stocks were titrated (7 (link)) for use in a PRNT adapted from a previously described method for SARS-CoV (36 (link)). Briefly, serological specimens were diluted 2-fold from 1:20 to 1:640 in DMEM supplemented with 2% FBS and incubated with 50 PFU of SARS-CoV-2 at 37°C and 5% CO₂ for 1 hour. The sera-virus mixtures were added to 12-well plates containing Vero E6 cells at 100% confluence, followed by incubation at 37°C and 5% CO₂ for 1 hour. After adsorption, a liquid overlay composed of 1.5% carboxymethylcellulose diluted in MEM, supplemented with 4% FBS, L-glutamine, nonessential amino acids, and sodium bicarbonate, was added to each well; the plates were incubated at 37°C and 5% CO₂ for 72 hours. The liquid overlay was removed, and the cells were fixed with 10% neutral-buffered formalin for 1 hour at room temperature. The monolayers were stained with 0.5% crystal violet for 10 minutes and washed with 20% ethanol. Plaques were enumerated and compared with controls. The highest serum dilution resulting in 50% and 90% reduction in plaques compared with controls were defined as the PRNT50 and PRNT90 endpoint titers, respectively. PRNT50 titers ≥ 1:160 and PRNT90 titers ≥ 1:20 were considered positive.
For the PRNT assay at Wadsworth, the assay for the detection of SARS-CoV-2 neutralizing antibodies was a modified version of previously described methods (37 (link)–39 (link)). Patient sera and SARS-CoV-2 (USA/WA-1/2020, BEI Resources, NR-52281) were diluted in Vero E6 cell culture maintenance medium (EMEM, 2% heat-inactivated FBS, 200 U/mL penicillin G, 200 U/mL streptomycin). Patient samples were serially diluted 1:10–1:320 and mixed with an equal volume of virus containing 150 PFUs. Virus and serum mixtures were incubated at 37°C and 5% CO₂ for 1 hour. Following the initial incubation, 0.1 mL of each dilution was plated in a single well of a 6-well plate containing confluent monolayers of Vero E6 cells (ATCC, CRL-1586) and allowed to adsorb for 1 hour at 37°C and 5% CO₂. Following adsorption, cell cultures were overlaid with 0.6% agar in cell culture medium and returned to the incubator. At 2 days after infection, a second overlay containing 0.2% neutral red was added. Monolayers were inspected for 2 days, and plaques were counted. Antibody titers were reported as the inverse of the serum dilution resulting in 50% (PRNT50) and 90% (PRNT90) reduction in plaques as compared with the virus inoculum control.
For the cytopathic effect–reduction neutralization assay in Toronto, 200 μL of 0.2 × 10⁶ VeroE6 cells/mL were seeded into a 96-well flat-bottom plate to adhere overnight. All plasma and serum samples were heat inactivated at 56°C for 30 minutes. In a separate 96-well plate, the serum, plasma, or antibody (1 μg/mL) samples were serially diluted 2-fold 8 times in serum-free DMEM starting from a dilution of 1:20 to 1:2560 in a volume of 25 μL. To all wells, 25 μL of SARS-CoV-2 SB2 Clone 1 was added, ensuring that each well had a dose of 100 issue culture infectious dose (TCID). For the cell control, 50 μL of serum-free DMEM was added. For the virus control, 25 μL of SARS-CoV-2 SB2 Clone 1 was added with a dose of 100 TCID and topped off with 25 μL of serum free DMEM. The plate was incubated for 1 hour at 37°C, 5% CO₂ with shaking every 15 minutes. After incubation, all the media from the VeroE6 culture were removed, and the full 50 μL of serum/SARS-CoV-2 coculture was layered on the cells. The plate was again incubated for 1 hour at 37°C, 5% CO₂, with shaking every 15 minutes. After the incubation, the inoculum was removed, and 200 μL of DMEM containing 2% FBS was added. The plate was incubated for 5 days and cytopathic effect was tracked.

To test the susceptibility of C. albicans strains to killing by human neutrophils, blood was collected from healthy volunteers by venipuncture and mixed with K₃EDTA (#E-0270, Sigma-Aldrich). The donors were two females and one male. The neutrophils were isolated using Lympholyte-Poly Cell Separation Media (#CL5070, Cedarlane) following the manufacturer’s instructions. They were washed once with HBSS without Ca⁺/Mg⁺ (#21–022-CV, Corning), suspended in RPMI 1640 medium with L-glutamine (#9161, Irvine Scientific) containing 10% pooled human serum (#100–110, Gemini Bioproducts, Inc.) and enumerated using a hemacytometer. For the killing assay, the neutrophils were incubated with C. albicans yeast at the ratio of 1:1 in polypropylene tubes at 37°C. As a control, an equal number C. albicans cells was incubated without neutrophils in parallel. After 3 h, the neutrophils were lysed by adding sterile water to the tube, followed by sonication. The number of viable organisms was determined by quantitative culture.
The susceptibility of the C. albicans strains to killing by mouse neutrophils was determined similarly. Neutrophils were purified from bone marrow cells using negative magnetic bead selection (MojoSort, BioLegend) as we have done before (Swidergall et al., 2019 (link)). In brief, bone marrow cells from a male and a female mouse were flushed from femurs and tibias using sterile RPMI 1640 medium supplemented with 10% FBS and 2 mM EDTA. After washing the cells with 1X MojoSort buffer (1X PBS, 0.5% BSA, 2mM EDTA), the neutrophils were isolated according to the manufacturer’s instructions. These neutrophils had > 95% purity and > 90% viability as determined by flow cytometry. For the killing assay, the mouse neutrophils were incubated with C. albicans yeast at the ratio of 1:20 in RPMI 1640 medium containing 2% heat-inactivated mouse serum (#S3509, Sigma-Aldrich) at 37°C for 3 h.