Anti c myc fitc

The yeast library was incubated with 125 nM of tetrameric SARS-CoV-1 and 1 µl of anti-c-myc FITC (Miltenyi Biotec) for 1 hour. Samples were then washed 2× with PBSM and resuspended in 50 µl of PBSM. These libraries were then sorted on a FACSAria IIu using the Stanford FACS Facility. The samples were gated such that all antigen-positive cells were collected (gates set such that ~0.5% anti-c-myc FITC alone controls fell within the gate). Two populations were sorted: a hi-gate, consisting of the highest intensity binders (3.8% of all cells), and a low-gate, consisting of all other antigen-positive cells (3.7% of all cells). Cells were sorted directly into tubes containing 4 ml of SD-CAA media. These sorted libraries were grown for 1 day at 30 °C shaking in SD-CAA media, and then 300 µl of the cultures were mini-prepped (Zymo Research), following the manufacturer’s protocol. mini-prepped DNA was transformed into STELLAR Competent Cells (Clontech) and plated on carbenicillin LB agar plates (as per pPNL6ʼs resistance marker). Escherichia coli cells that grow should, theoretically, contain only a single sequence from each of the yeast that were sorted above. Ten E. coli colonies from the hi-gate and 20 E. coli colonies from the low-gate sort were sent for sequencing (Sequetech). The sequences were then analyzed by sequence alignment using SnapGene software (version 6.0.2).

After overnight induction, yeast cultures were pelleted, washed twice with 0.01% PBSA (VWR #45001–130; GoldBio, St. Louis, MO, #A-420–50), and resuspended to an OD600 of 1. A total of 500-700 μL of OD1 yeast cells were labeled with biotinylated human ACE2 (Acrobiosystems #AC2-H2H82E6) at each of the twelve ACE2 concentrations (half-log increments spanning 10^−12.5–10⁻⁷M), with volumes adjusted to limit ligand depletion effects to be less than 10% (assuming 50,000 surface RBD/cell^{37 (link)}). Yeast-ACE2 mixtures were incubated and rotated at room temperature for 20 hr. Following the incubation, yeast-ACE2 complexes were pelleted by spinning at 3000 × g for 10 min at 4 °C, washed twice with 0.5% PBSA + 2 mM EDTA, and subsequently labeled with Streptavidin-RPE (1:100, Thermo Fisher #S866) and anti-cMyc-FITC (1:50, Miltenyi Biotec, Somerville, MA, #130-116-485) at 4 °C for 45 min. After this secondary labeling, yeast were washed twice with 0.5% PBSA + 2 mM EDTA and left on ice in the dark until sorting.

For flow cytometry, ~1 × 10⁶ induced cells were washed with 200 µl PBS/0.5% BSA (30 sec spin in a benchtop minicentrifuge, ~1000 × g) and incubated with 2.5 µM A2M substrate in a 50 µl volume at room temperature on a roller. Cells were then washed three times as was the case for the subsequent labelling steps. Streptavidin-PE (1:200, Biolegend, cat. no. 405204) was first applied for 1 h followed by staining with PE anti-streptavidin (1:200, Biolegend, cat. no. 410503, clone 3A20.2) and anti-c-myc-FITC (1:50, Miltenyi Biotec, cat. no. 130-116-485, clone SH1-26E7.1.3) also for 1 h. Flow cytometry was carried out on a Becton Dickinson FACScan Cytek DxP machine with a 488 nm laser / 530/30 nm filter for FITC and a 561 nm laser / 590/20 nm filter for PE. Flow cytometry data was analysed with FlowJo v10.1.

On day 4, yeast-HA complexes were pelleted by spinning at 3000 x g for 10 min at 4°C, washed twice with 5% PBSA + 2 mM EDTA, and simultaneously labeled with Streptavidin-RPE (1:100, Thermo Fisher #S866) and anti-cMyc-FITC (1:50, Miltenyi Biotec, Somerville, MA, #130-116-485) at 4°C for 45 min. Following secondary labeling, yeast were washed twice with 5% PBSA + 2 mM EDTA, and left on ice in the dark until sorting.

Cells were collected from culture and incubated with MIL-38-CD3 (10 μg/ml) for 45 min on ice. Cells were washed 3 times with PBS/2% HI FBS (Flow cytometry staining wash; FSW) then stained with anti-cmyc-FITC (Miltenyi Biotec, Australia; clone SH1-26E7.1.3) or anti-his FITC or PE (Miltenyi Biotec, Australia; clone GG11-8F3.5.1) for 45 min on ice. Cells were washed 3 times in FSW and then acquired on the BD Fortessa X20 (BD, Australia) using FACS DIVA software (BD, Australia). The viability dye topro-3-iodide (Life Technologies; Australia) was used to restrict analysis to viable cells. This dye was added to a final concentration of 1 μM immediately prior to acquisition. Data were analysed in FCS Express V5 (De Novo Software, USA).