Vero cells (1.2 × 104 cells) were cultured in DMEM with 1× antibiotic–antimycotic solution and 2% FBS in 384-well black culture plates. The serially diluted compounds and 0.0125 multiplicity of infection (MOI) SARS-CoV-2, 0.05 MOI SARS-CoV, or 0.0625 MOI MERS-CoV were added. At 24 h post-infection, the cells were fixed with 4% paraformaldehyde and stained with antibodies against the SARS-CoV-2 nucleocapsid protein, SARS-CoV spike protein, or MERS-CoV spike protein (Sino Biological Inc., Beijing, China), followed by goat antirabbit IgG secondary antibody and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). Images were analyzed using the Operetta® High-Content Imaging System (20×; PerkinElmer, Inc., Waltham, MA, USA) and Image-Mining 3.0 plug-in software [20 (link)].
Sars cov 2 nucleocapsid protein
Manufactured by Sino Biological
Sourced in China
The SARS-CoV-2 nucleocapsid protein is a structural protein that forms the viral capsid. It plays a crucial role in the viral genome packaging and assembly.
Automatically generated - may contain errors
Lab products found in correlation
Sars cov spike protein, by Sino Biological (1 mentions)
Mers cov spike protein, by Sino Biological (1 mentions)
Operetta high content imaging system, by PerkinElmer (1 mentions)
S1pr2, by Proteintech (1 mentions)
S1pr2, by BioGenex (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti s1, by Sino Biological (1 mentions)
Sars cov 2 spike protein s1 s2, by Sino Biological (1 mentions)
Sars cov 2 spike rbd protein, by Sino Biological (1 mentions)
7 protocols using sars cov 2 nucleocapsid protein
1
High-Content Screening of SARS Inhibitors
2
Histological Evaluation of Lung Tissue
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5 micron sections were stained with hematoxylin and eosin (H&E) to evaluate tissue morphology and air space and Masson’s trichrome stain to evaluate degree of collagen deposition. Immunohistochemistry was performed to detect SK1 (1:200, LS Bio, Seattle, WA, USA), CERS2 (1:1200, OTI3D9, OriGene, Rockville, MD, USA), S1PR1 (1:250, 8B7.1, MilliporeSigma, Burlington, MA, USA), or S1PR2 (1:200, Proteintech, Rosemont, IL, USA) using the Dako Envision + Dual Link secondary detection kit (Agilent Technologies, Santa Clara, CA) and chromogens 3,3’-Diaminobenzidine (DAB; brown; SK1, CERS2, and S1PR1 staining) or AEC (3-amino-9-ethylcarbazole) HRP Substrate (red; S1PR2 staining) following antigen retrieval (SK1, S1PR1 & S1PR2:10 mM citrate buffer (pH 6, Biogenex, San Ramon, CA, USA), and CERS2: Tris/EDTA buffer (pH9, Agilent, Santa Clara, CA, USA)). Representative no antibody staining controls shown in Supplemental Figure 2
3
COVID-19 Infection Monitoring in Vaccine Trial
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At each time point/visit, patients were asked whether they had been infected with COVID-19 (validated by PCR) and, if so, whether they had any symptoms (i.e. abdominal pain, diarrhoea, nausea, vomiting, fever, coughing, shortness of breath, fatigue, and myalgia) and whether hospitalisation with/without oxygen supply was needed. This information was recorded in the HEPCOVIVac Registry on REDCap™. At each time point/visit, previous COVID-19 infection was confirmed or evaluated for all patients by testing for the SARS-CoV-2 anti-nucleocapsid protein antibody. Briefly, the SARS-CoV-2 nucleocapsid protein (Sino Biologicals) was used in conjugation with a goat anti-human IgG Fc horseradish peroxidase-conjugated antibody (Abcam) in an ELISA. Patients who developed infection before T2 (n = 75) were used to query for associations with demographic or clinical characteristics, whereas patients who developed infection between T2 and T3 (n = 29) were used to measure vaccine efficacy.
4
SARS-CoV-2 Antibody Detection ELISA Assay
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In this study, two in-house enzyme-linked immunosorbent assay (ELISA) systems were used to assess the participants’ anti-spike IgG (anti-S1 + RBD IgG) and anti-N IgG antibody levels [17 (link),18 (link),19 (link)]. Briefly, ELISA plates were pre-coated with SARS-CoV-2 Spike 1 and RBD proteins (Sino biological, Beijing, China) at a ratio of 6:4, and SARS-CoV-2 nucleocapsid protein (Sino biological, Beijing, China) for anti-spike IgG (anti-S1 + RBD IgG) and anti-N IgG detection. After the serum separation, the serum was diluted (1:100) with dilution buffer and dispensed into wells with positive, negative, and plate controls. After incubation for 15 min and a washing step, horseradish peroxidase-conjugated anti-human IgG (The NativeAntigens, London, UK) at a 1:4000 dilution was added to the wells. After a short incubation and wash, the substrate, 3,3′,5,5′-Tetramethylbenzidine (TMB), was added to each well, followed by a stop solution. The optical density of the final reaction was measured at 450 nm. The antibody level was finally determined by analyzing the OD/cut-off ratio.
5
Histological Assessment of SARS-CoV-2 Lung Infection
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Lung tissues of SARS-CoV-2-infected animals were collected and fixed in 4% paraformaldehyde for 3 days, followed by embedding in paraffin and cutting into 3 μm sections. Some sections were stained with H&E for evaluation of lung injury, and other sections were stained with the SARS-CoV-2 nucleocapsid protein (Sino Biological) to evaluate SARS-CoV-2 replication levels in the lung tissues.
6
SARS-CoV-2 Recombinant Protein Evaluation
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SARS-CoV-2 Spike Protein (S1+S2) (cat# 40589-VO8B1), SARS-CoV-2 Spike RBD Protein (cat# 40592-V08B), SARS-CoV-2 Nucleocapsid Protein (cat# 40588-V08B) were purchased from Sino Biological (China).
7
SARS-CoV-2 Nucleocapsid Protein IFA
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Cells were fixed with 4 % paraformaldehyde for 30 min, followed by two PBS washes and permeabilization with 0.125% Triton X-100 in PBS for 30 min. After blocking in 2% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological) at a 1:3000 dilution for 1h followerd by incubation with a secondary AlexaFluor647-labeled antibody. Nuclei were stained using Hoechst33342. Single images were acquired using an Echo Revolve inverted fluorescence microscope. IFA staining in whole wells was quantified using automated image acquisition, on a Cytation 5 Cell Imaging reader with 12 images per well to cover the complete well. Nuclei and AlexaFluor647-positive cells were counted using the manufacturer's provided software. After subtraction of background (uninfected) controls, all signals were normalized to the mock transfected controls.
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