Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation and cryopreserved. SARS-CoV-2 recombinant protein was generated, briefly SARS-CoV-2 Spike RBD domain consisted of residues Thr-333 to Thr-531 (uniprot P0DT2). The RBD sequence was cloned in frame with a his8 and avi-tag sequence, respectively (RBDhisavi). RBDhisavi was expressed in insect cells and purified by nickel affinity chromatography. Purified RBDhisavi was biotinylated in vitro using BIRA (https://www.avidity.com/ ) for subsequent formation of RBDhisavi-tetramers for B cell staining experiments. The same RBD sequence was cloned in frame with a murine IgG1 FC domain (RBD-FC). RBD-FC was expressed in insect cells and purified by protein A affinity chromatography. Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described,44 (link) using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).
Af933 sp
Manufactured by R&D Systems
AF933-SP is a lab equipment product manufactured by R&D Systems. It is a device designed for specific laboratory applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. More information may be available from the manufacturer.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 percp cy5, by BioLegend (1 mentions)
Anti cd19 apc cy7 sj25c1, by BD (1 mentions)
Cd4 bv510, by BioLegend (1 mentions)
Cd3 bv510 okt3, by BioLegend (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Igd fitc ia6 2, by BD (1 mentions)
Aurora flow cytometer, by Cytek (1 mentions)
3 protocols using af933 sp
1
SARS-CoV-2 Spike RBD Protein Characterization
2
SARS-CoV-2 Infection in ACE2-Expressing Cell Lines
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HEK293T-ACE2,
A549-ACE2, and H23-ACE2 stable cell lines and SARS-CoV-2 infection
were performed as described previously.63 (link) SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020; GISAID accession no.
EPI_ISL_425177) was kindly provided by Darryl Falzarano (Vaccine and
Infectious Disease Organization, Saskatoon, Canada). HEK 293T-ACE2
and A549-ACE2 cells were developed by electroporating a human ACE2
encoding plasmid (Addgene #1786; a gift from Hyeryun Choe). The cells
were passaged six times in culture, surface-stained for ACE2 (goat
anti-ACE2; AF933-SP; R&D Systems), and the highest 2% of cells
expressing ACE2 were sorted from the bulk population. Virus culture
and experiments were performed according to level-3 containment procedures.
Virus stocks were generated and titrated (by plaque assay) in Vero
E6 cells, and HEK293T-ACE2, A549-ACE2, and H23-ACE2 cells were infected
using MOI = 1.
A549-ACE2, and H23-ACE2 stable cell lines and SARS-CoV-2 infection
were performed as described previously.63 (link) SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020; GISAID accession no.
EPI_ISL_425177) was kindly provided by Darryl Falzarano (Vaccine and
Infectious Disease Organization, Saskatoon, Canada). HEK 293T-ACE2
and A549-ACE2 cells were developed by electroporating a human ACE2
encoding plasmid (Addgene #1786; a gift from Hyeryun Choe). The cells
were passaged six times in culture, surface-stained for ACE2 (goat
anti-ACE2; AF933-SP; R&D Systems), and the highest 2% of cells
expressing ACE2 were sorted from the bulk population. Virus culture
and experiments were performed according to level-3 containment procedures.
Virus stocks were generated and titrated (by plaque assay) in Vero
E6 cells, and HEK293T-ACE2, A549-ACE2, and H23-ACE2 cells were infected
using MOI = 1.
3
SARS-CoV-2 Spike Protein Binding Assay
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Ten microliters of diluted heat-inactivated serum was incubated with an equal volume of spike protein–coated fluorescent beads for 2 hours at 37°C, 5% CO2 to allow immune complexes to form. THP-1 (ATCC, TIB-202) cells were then added (5 × 104 cells per well in a volume of 200 μl) and incubated for 16 hours at 37°C, 5% CO2. One-hundred microliters of supernatant was removed from each well and replaced with an equal volume of 4% paraformaldehyde and incubated for 20 min at RT. Cells were pelleted and washed once in fluorescence-activated cell sorting (FACS) buffer (10% fetal bovine serum and 0.5 mM EDTA in PBS), and percentage of THP-1 cells with internalized spike protein beads was quantified using a Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, CA) and analyzed in FlowJo (BD Biosciences, Ashland, OR). Human Fc receptor–blocking agent (BD Pharmingen, 564220; 2 μg per well) and anti-human ACE-2 R antibody [R&D, AF933-SP; final concentration (2 μg/ml)] were added to THP-1 cells as additional controls.
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