Anti human cd3 af700 clone ucht1

Biotinylated SARS-CoV-2 spike proteins from D614G and BA.1 variants were incubated with yellow-green streptavidin-fluorescent beads (Molecular Probes) for 2 hours at 37°C. Ten microliters of a 100-fold dilution of beads–protein was incubated for 2 hours at 37°C with 100 μl of a 1000-fold diluted plasma samples before the addition of effector cells (50,000 cells per well). Fresh peripheral blood leukocytes from human were used as effector cells after red blood cell lysis with Ammonium-Chloride-Potassium lysing buffer (Thermo Fisher Scientific). After 1 hour of incubation at 37°C, the cells were washed, surface-stained, and fixed with 4% formaldehyde solution (Tousimis, Rockville, MD, USA), and fluorescence was evaluated on an LSR II (BD Biosciences). Antibodies used for flow cytometry were anti-human CD3 AF700 (clone UCHT1) and anti-human CD14 APC-Cy7 (clone MϕP9; both from BD Biosciences) and anti-human CD66b Pacific Blue (clone G10F5, BioLegend). The ADNP phagocytic score was calculated by multiplying the percentage of bead-positive neutrophils (side-scatter (SSC) high, CD3⁻CD14⁻CD66⁺) by the geometric MFI of the bead-positive cells and dividing the product by 10⁴.

Biotinylated SARS-CoV-2 prefusion stabilized S trimer was incubated with yellow-green streptavidin-fluorescent beads (Molecular Probes) for 2 h at 37 °C. Ten microliters of a 100-fold dilution of protein-coated beads was incubated for 2 h at 37 °C with 100 μL of 8,100-fold diluted plasma samples before addition of effector cells (50,000 cells per well). Fresh human PBMCs were used as effector cells after red blood cell lysis with Ammonium-Chloride-Potassium (ACK) lysing buffer (ThermoFisher Scientific). After 1 h incubation at 37 °C, cells were washed, surface stained, and fixed with 4% formaldehyde solution (Tousimis), and fluorescence was evaluated on an LSRII flow cytometer (BD Bioscience). Antibodies used for flow cytometry included anti-human CD3 AF700 (clone UCHT1), anti-human CD14 APC-Cy7 (clone MϕP9) (BD Bioscience, San Jose, C), and anti-human CD66b Pacific Blue (clone G10F5) (BioLegend). The phagocytic score was calculated by multiplying the percentage of bead-positive neutrophils (SSC high, CD3− CD14− CD66+) by the geometric mean of the fluorescence intensity of bead-positive cells and dividing by 10,000.

Biotinylated SARS-CoV-2 stabilized trimer was incubated with yellow-green streptavidin fluorescence beads (Molecular Probes) for 2 h at 37 °C. Next, 10 μl of a 100-fold dilution of beads–protein mixture was incubated with mAbs as described above before addition of effector cells (50,000 cells per well). Fresh peripheral blood leukocytes from human samples were used as effector cells after red blood cell lysis with ACK lysing buffer (Thermo Fisher Scientific). After 1 h of incubation at 37 °C, the cells were washed, surface stained, fixed with 4% formaldehyde solution and fluorescence was evaluated on an LSR II (BD Bioscience). Antibodies used for flow cytometry were anti-human CD3 AF700 (clone UCHT1) and anti-human CD14 APC-Cy7 (clone MϕP9; both BD Biosciences) and anti-human CD66b Pacific Blue (clone G10F5, BioLegend). The phagocytic score was calculated by multiplying the percentage of bead-positive neutrophils (SSC^hiCD3⁻CD14⁻CD66⁺) by the geometric MFI of the bead-positive cells and dividing by 10⁴.