Assays to measure spike-specific ADCP were performed using a protocol reported previously (25 (link), 27 (link)). Briefly, FluoSpheres carboxylate-modified yellow-green fluorescent microspheres (Thermo Fisher, #F8823) were coupled with SARS-CoV-2 spike or RBD proteins using the xMAP Antibody Coupling Kit (5 µg protein/~36.4x109 beads, Luminex #40-50016). Spike-conjugated microspheres were incubated with diluted plasma for 2 hours at 37°C in the dark. After washing and centrifugation (2,000 g, 10 min), the beads (~3x108 beads, 10 µL/well) were incubated with THP-1 cells (0.25x105 cells, 200 µL/well) for 16 hours. The samples were analyzed on an Attune NxT flow cytometer (Thermo Fisher, #A24858). Data analysis was performed using FCS Express 7 Research Edition (De Novo Software). ADCP scores were calculated as follows: (% microsphere positive cells) x (geometric mean fluorescent intensity of the microsphere positive cells)/1000.
Fluospheres carboxylate modified yellow green fluorescent microspheres
Manufactured by Thermo Fisher Scientific
FluoSpheres carboxylate-modified yellow-green fluorescent microspheres are fluorescent particles designed for use as a tracer, label, or reference standard in various applications. The microspheres are composed of a polystyrene matrix and contain a yellow-green fluorescent dye. The carboxylate modification provides surface functionality for attachment of biomolecules.
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2 protocols using fluospheres carboxylate modified yellow green fluorescent microspheres
1
Measuring SARS-CoV-2 Spike ADCP Activity
2
Spike-specific ADCP Measurement Assay
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Assays to measure spike-specific ADCP were performed using a published protocol83 (link) with some modifications reported elsewhere.82 (link) Briefly, FluoSpheres carboxylate-modified yellow-green fluorescent microspheres (Thermo Fisher, #F8823) were coupled with SARS-CoV-2 spike protein using the xMAP Antibody Coupling Kit (5 μg protein/∼36.4 × 109 beads, Luminex #40–50016). Spike-conjugated microspheres were incubated with diluted plasma for 2 h at 37°C in the dark. After washing and centrifugation (2,000 g, 10 min), the beads (∼3 × 108 beads, 10 μL/well) were incubated with THP-1 cells (0.25 × 105 cells, 200 μL/well) for 16 h. The samples were analyzed on an Attune NxT flow cytometer (Thermo Fisher, #A24858). Data analysis was performed using FCS Express 7 Research Edition (De Novo Software). ADCP scores were calculated as follows:
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