96 deep well plate

Individual colonies, selected from the third or fourth round of biopanning, were inoculated into 1 mL of SB media supplemented with 50 μg/mL of carbenicillin in 96-deep-well plates (Axygen, Union City, CA, USA) and incubated at 37 °C for 5 h. Then, 10¹¹ pfu/mL of helper phages (VCSM13) and 70 μg/mL of kanamycin were added to the plates and incubated overnight at 37 °C. The plates were centrifuged at 3000× g for 30 min, and the phage supernatant was used for ELISA. Ninety-six-well high-binding microplates (Corning, NY, USA) were coated with 0.1 μg recombinant SARS-CoV-2 BA.2 RBD and rhCD155 (Sino Biological) and incubated overnight at 4 °C. After blocking using 3% (w/v) BSA in phosphate-buffered saline (PBS), the plates were incubated with 100 μL of phage supernatant at 37 °C for 2 h. After washing thrice with PBS containing 0.05% (v/v) Tween 20 (PBST), HRP-conjugated anti-hemagglutinin antibody (1:3000; Bethyl Laboratories, Montgomery, TX, USA) was added and incubated at 37 °C for 1 h. Colorimetric detection was performed by using the TMB substrate solution (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was terminated by the addition of 1 M H₂SO₄, and absorbance was measured at 450 nm using a microtiter plate reader (Bio-Tek Instruments).

Single colonies were inoculated into 1 mL of SB media containing 50 μg/mL of carbenicillin in 96-deep-well plates (Axygen, Union City, CA, USA) and incubated at 37 °C overnight. Then, 10¹² pfu/mL helper phages (VCSM13; Agilent, Santa Clara, CA, USA) and 70 μg/mL of kanamycin were added and incubated overnight at 37 °C. The plates were centrifuged at 4000 rpm, and the supernatant was applied for phage ELISA. The 96-well high-binding microplates (Corning, Corning, NY, USA) were coated overnight with 0.1 μg of MF-hCADM1 and MF-mCADM1 in phosphate-buffered saline (PBS) at 4 °C. After blocking with 3% (w/v) BSA in PBS, the plates were incubated with 100 μL of phage supernatant at 37 °C for 2 h. After washing three times with PBS containing 0.05% (v/v) tween 20 (PBST), HRP-conjugated anti-hemagglutinin (HA) antibody (1:3000; Bethyl Laboratories, Montgomery, TX, US) was added and incubated at 37 °C for 1 h. Colorimetric detection was performed by adding 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) as the chromogenic substrate. The reactions were stopped by the addition of 2 M of sulfuric acid (H₂SO₄), and the absorbance was read at 450 nm using a microtiter plate reader (Bio-Tek Instruments, Winooski, VT, USA).

In total, 50 µL of 30× concentrated (not purified) VLPs were preincubated for 45–60 min at RT with 50 µL of antibodies or sera or PBS at the indicated concentration or dilution. During that time, transfected Expi293F cells expressing ACE2 or a mock control were counted and 0.5 × 10⁶ cells per well were prepared in 400 µL of FACS buffer (2% FCS, 10 mM EDTA, 1× PBS, pH 7.4) in a 96-deep-well plate (Axygen, Corning, CA, USA). After centrifugation for 4 min at 280× g, the supernatant was removed, and the cells were resuspended in the 100 µL of VLP mix and incubated at 37 °C for 60 min before 400 µL of FACS buffer was added. The plate was centrifuged again for 4 min at 280× g and supernatant was removed. This was followed by a washing step, resuspending the cells in 400 µL of FACS buffer and centrifugation for 4 min at 280× g. The resulting cell pellet was resuspended in 200 µL of FACS buffer and measured in a flow cytometer (MACSQuant, Miltenyi Biotech, Bergisch Gladbach, Germany or BD FACSMelody, BD Biosciences, Franklin Lakes, NJ, USA). Analysis was performed by Flowing Software 2 (Turku Bioscience Centre, Turku, Finland) or FlowJo 10.8.1 (BD Biosciences, Franklin Lakes, NJ, USA) gating single cells and measurement of their GFP median (see Supplementary Figure S2for the gating strategy).

DNA isolation from dislodged biofilms was performed as described previously [41 (link)]. Briefly, biofilm pellets were thawed, resuspended with 150 μl Tris EDTA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0)) and transferred to a well in a 96 deep well plate (Axygen Scientific Inc., CA, USA). As controls, the original inoculum and sterile McBain medium without additions from each experiment were included.
To each well, the following compounds were added: 250 μl of 0.1-mm diameter Zirconia beads (BioSpec Products, Bartlesville, OK, USA), 200 μl of phenol (Rotiphenol, Carl Roth GMBH&Co. KG, Germany) and 200 μl of lysis buffer (MagMini DNA isolation kit, LGC Genomics Ltd, UK). The plate was then sealed and placed in a Mini-BeadBeater-96 (BioSpec Products, Bartlesville, OK, USA) for 2 min at 2.100 oscillations/min. When this process was completed, DNA was extracted and purified with the MagMini DNA Isolation Kit (MagMini DNA isolation kit, LGC Genomics Ltd, UK). Bacterial DNA concentration was determined by qPCR as described elsewhere [42 (link)].