Detection of SARS-CoV-2-specific memory B cells (MBCs) by flow cytometry was as previously described.21 (link) In brief, for SARS-CoV-2 specific MBCs responses, biotinylated SARS-CoV-2 Spike RBD protein (40592-V08H2-B; Sino Biological, Beijing, China) was mixed with Streptavidin BV421 (405225; Biolegend, San Diego, CA, USA) at 4:1 molar ratio for 1 h at 4°C to obtain the antigen probe. According to the manufacturer’s instruction, peripheral blood mononuclear cells (PBMCs) were stained for 30 m at 4°C using antigen probe (1:33.3) and the following conjugated antibodies: antihuman CD3 (1:50) (300430; Biolegend), antihuman CD19 (1:50) (302212; Biolegend), antihuman CD21 (1:50) (354918; Biolegend), antihuman CD27 (1:50) (356406; Biolegend). After staining, cells were rewashed and resuspended in a 200ul FACS buffer. Samples were then evaluated by flow cytometry (CytoFLEX; Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo (version 10.0.7r2; Treestar, Woodburn, OR, USA).
Anti human cd27
Manufactured by BioLegend
Sourced in United States
Anti-human CD27 is a monoclonal antibody that recognizes the CD27 cell surface antigen expressed on T cells, B cells, and natural killer cells. CD27 is a member of the tumor necrosis factor receptor superfamily and is involved in the activation and proliferation of lymphocytes.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin bv421, by BioLegend (5 mentions)
Anti human cd19, by BioLegend (5 mentions)
Anti human cd21, by BioLegend (5 mentions)
Anti human cd3, by BioLegend (4 mentions)
Histopaque, by Merck Group (3 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Anti human igm, by BioLegend (2 mentions)
Histopaque density gradient centrifugation, by Merck Group (1 mentions)
Beckman flow cytometer, by Beckman Coulter (1 mentions)
40592 v08h2 b, by Sino Biological (1 mentions)
Beckman flow cytometry, by Beckman Coulter (1 mentions)
5 protocols using anti human cd27
1
SARS-CoV-2 Memory B Cell Detection
2
SARS-CoV-2 Spike RBD Protein Binding Assay
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Streptavidin BV421 (405225; Biolegend, California, CA, USA) and biotinylated SARS-CoV-2 Spike RBD protein ((40592-V08H2-B; Sino Biological, Beijing, China) were incubated for 1 hour at 4°C to create the antigen probe. Peripheral blood mononuclear cells (PBMCs) were separated from heparinized blood base on the manufacturer’s recommendations using Histopaque density gradient centrifugation (10771, Sigma-Aldrich). After rinsing with FACS buffer (phosphate-buffered saline with 2% fetal bovine serum), staining was performed for 30 min at 4°C with an antigen probe (1:33.3) and the following binding antibodies: anti-human CD3 (300430, Biolegend), anti-human CD19 (302212, Biolegend), anti-human CD21 (354918, Biolegend), and anti-human CD27 (356406, Biolegend). These antibodies were added in 1:50 volume. After staining, cells were rinsed and resuspended in 150 µL FACS buffer. Subsequently, samples were evaluated using a flow cytometer (CytoFLEX, Beckman Coulter). FlowJo software version 10.0.7r2 was used for data analysis.
3
SARS-CoV-2 Spike Protein Antibody Detection
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The stained peripheral blood mononuclear cells were evaluated in the following procedure, using a Beckman flow cytometer (Beckman Coulter, Inc., California, USA). Antigen probe was obtained, and the biotinylated SARS-CoV-2 Spike RBD protein (40592-V08H2-B, Sino Biological, Beijing, China) was mixed with Streptavidin-BV421 (405225, Biolegend, California, USA) at a 4:1 molar ratio for 1 h. To obtain the peripheral blood mononuclear cells, density gradient centrifugation [Histopaque (10771, Sigma-Aldrich, St Louis, Missouri, USA)], cell cleaning (FACS, With 2% FBS), add antigen probes (1:33.3) and fluorescent-conjugated antibodies [anti-human IgG Fc (410722, Biolegend), anti-human IgM (314524, Biolegend) Antihuman CD3 (300430, Biolegend), anti-human CD19 (302212, Biolegend), anti-human CD21 (354918, Biolegend), anti-human CD27 (356406, Biolegend)] after being mixed and placed at 4°C, 30 min, conduct light-avoidance staining; Test sample (FACS buffer), Analyze the data [FlowJo software (V10.0.7)].
4
SARS-CoV-2 Spike RBD Protein Binding Assay
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The stained peripheral blood mononuclear cells were tested by Beckman flow cytometry (Beckman Coulter, Inc., California, USA). The specific steps were as follows: first, we mixed the biotinylated SARS-CoV-2 spike RBD protein (40592-V08H2-B, Sino biological, Beijing, China) with streptavidin-BV421 (405225, Biolegend, California, USA) in a 4:1 mole ratio and leave for 1 h to get the antigen probe; second, peripheral blood mononuclear cells were obtained from whole heparinized blood; the density gradient centrifugation was performed by Histopaque (10771, Sigma–Aldrich, St Louis, Missouri, USA) and then cleaned by cytometric staining buffer (FACS, 2%FBS) cells, besides we added antigen probe (1:33.3), fluorescence-coupled antibody [anti-human IgG Fc (410722, Biolegend), anti-human IgM (314524, Anti-human) CD3 (300430, Biolegend), anti-human CD19 (302212, Biolegend), anti-human CD21 (354918, Biolegend), and anti-human CD27 (356406, Biolegend)] into the cells and dyed at 4°C for 30 min under dark condition. After being re-suspended with FACS buffer, the samples were tested on the machine. The data were analyzed by Flow Jo software (V10.0.7).
5
Quantifying SARS-CoV-2 Spike Antibodies
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Biotinylated SARS-CoV-2 spike RBD protein (Sino Biological, 40592-V08H2-B) was mixed with streptavidin BV421 (Biolegend, 405225) at a 4:1 molar ratio for 1 hour at 4°C to generate the antigen probe. According to the manufacturer's instructions, peripheral blood mononuclear cells (PBMCs) were extracted from heparinized whole blood by density gradient centrifugation with Histopaque (Sigma–Aldrich, 10771). After rinsing with FACS buffer (PBS+2% FBS), PBMCs were stained at 4°C for 30 minutes with an antigen probe (1:33.3), and the subsequent conjugated antibodies, namely anti-human CD3 (300430, Biolegend, 1:50), anti-human CD19 (302212, Biolegend, 1:50), anti-human CD21 (354918, Biolegend, 1:50), and anti-human CD27 (356406, Biolegend, 1:50). FACS cells were resuspended in 200 µm of FACS buffer after staining. The samples were subjected to flow cytometric evaluation (Beckman Coulter, CytoFLEX) and FlowJo analysis (Treestar, 10.0.7r2).
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