Cells were firstly incubated with Fc blocker (Human BD Fc Block™, #564219, BD Biosciences). Then they were washed and used incubation for reagents in two different settings: purified SARS-CoV-2 (2019-nCoV) Spike Protein (#40591-V02H, Sino Biological, China), whereas the other with rabbit anti-human ACE2 antibodies (#21115-1-AP, Proteintech, China) in DMEM (Gibico) supplemented with 10% FBS (Gibico) for 30 minutes at room temperature, followed by goat anti-rabbit IgG (Alexa Fluor 546 Goat anti-Rabbit IgG (H+L) polyclonal antibody, ThermoFisher) as secondary antibodies according to manufactures’ instructions. Subsequently, cells treated with the above mentioned reagents in two experimental settings were respectively labeled simultaneously by antibodies against human CD3 (APC mouse anti-human CD3 monoclonal antibody, clone HIT3a, BD Bioscience), CD14 (FITC mouse anti-human CD14 monoclonal antibody, clone RMO52, Beckman Coulter), CD68 (PE/Cy7 mouse anti-human CD68 monoclonal antibody, clone Y1/82A, Biolegend) and human IgG (PE anti-human IgG Fc, clone M1310G05, Biolegend). BD LSRFortessa™ X-20 was used for flow cytometry analysis.
2019 ncov spike protein
Manufactured by Sino Biological
Sourced in China
The 2019-nCoV Spike Protein is a recombinant protein that corresponds to the spike protein of the SARS-CoV-2 virus. The spike protein is a key structural component of the virus and plays a crucial role in the virus's ability to bind to and infect host cells.
Automatically generated - may contain errors
Lab products found in correlation
Human fc block, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Monensin, by Merck Group (1 mentions)
Anti cd3 okt3, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti mouse iga, by Abcam (1 mentions)
Costar elisa plate, by Corning (1 mentions)
Tmb 3 3 5 5 tetramethylbenzidine, by Beyotime (1 mentions)
Goat anti mouse igg, by ZSGB-BIO (1 mentions)
3 protocols using 2019 ncov spike protein
1
SARS-CoV-2 Spike Protein and ACE2 Binding
2
SARS-CoV-2 Spike Protein Stimulation of PBMCs
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Heparinized venous blood was collected from healthy donors. The PBMCs were isolated using Ficoll–Paque density gradient centrifugation. The PBMCs were seeded in a 96-well plate, approximately at 3 × 105 cells in 100 μl of RPMI per well. For activation, the cells were stimulated with anti-CD3 (OKT3, eBioscience, 10 μg/ml) and anti-CD28 (CD28.2, eBioscience, 10 μg/ml) and treated with SARS-CoV-2 (2019-nCoV) Spike protein (100 nM, Sino Biological) for 24 h. To measure the intracellular cytokine release, the cells were stimulated with PMA (50 ng/ml, Sigma-Aldrich) and ionomycin (1 μg/ml, Sigma-Aldrich) for 4–6 h in the presence of monensin (1 μg/ml, Sigma-Aldrich), collected and analyzed by flow cytometry.
3
ELISA Detection of SARS-CoV-2 Spike Antibodies
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2019-nCoV spike protein-specific IgA and IgG titres were detected with ELISA. Briefly, 0.2 μg 2019-nCoV spike protein (Sino Biological, Beijing, China), and B.1.1.529 spike protein (Sino Biological) were coated overnight onto the Costar ELISA plates (Corning, NY, USA), respectively. The 2019-nCoV spike protein was fused with a polyhistidine tag at the C-terminus, while the B.1.1.529 spike protein was fused with the bacteriophage T4 fibritin and a polyhistidine tag at the C-terminus. After blocking with 0.05% Tween 20-containing PBS and 1% bovine serum albumin at 37°C for 1 h, the plates were rinsed 6 times with 0.05% Tween 20-containing PBS, the diluted sera were added to the wells by 4-fold serial dilutions. After washing 6 times with 0.05% Tween 20-containing PBS, the plates were exposed to horseradish peroxidase-conjugated goat anti-mouse IgA (1:10,000; Abcam, UK) or goat anti-mouse IgG (1:10,000; ZSGB-BIO, China) at 37°C for 1 h. TMB (3,3’,5,5’-tetramethylbenzidine; Beyotime, China) was employed as a substrate to determine the antibody responses by measuring the absorbance at 450 and 630 nm. The end point titres were defined as the highest reciprocal serum dilution, which was 2.1-fold higher compared to the negative control.
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