PBMCs were isolated from whole blood collected from family members and healthy donors by ficoll-histopaque gradient centrifugation. For phenotypic staining, the following monoclonal antibodies were used: CD3-APC-H7, CD4-PerCP-Cy5.5, CD38-PerCP-Cy5.5, CD10-PECF594, CD21-APC, IgG PeCy7, CD14-PerCP, CD123-PE, CD56-PeCy7, CD11c-APC, CD16-APC-H7 (BD Biosciences, San Diego, CA, USA), CD8-APC-EF780, CD27-APC-EF780 (eBioscience, San Diego, CA, USA), CD19-BV650, CD24-BV605 (Biolegend, San Diego, CA, USA) and IgA-PE (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were enriched by negative selection using B-cell isolation kit (Stemcell, Vancouver, BC, Canada). Naïve B-cell purity was verified by flow cytometry to 98% purity.
Cd21 apc
Manufactured by BD
Sourced in United States
The CD21-APC is a fluorescently labeled antibody used in flow cytometry to identify and quantify CD21-expressing cells. CD21, also known as the complement receptor 2 (CR2), is a cell surface protein expressed on mature B cells, follicular dendritic cells, and some T cells. The APC (Allophycocyanin) fluorescent label allows for the detection and analysis of CD21-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd56 pe cy7, by BD (3 mentions)
Cd14 percp, by BD (2 mentions)
Cd19 percp, by BD (2 mentions)
Cd4 fitc, by BD (2 mentions)
Cd4 percp, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Cd4 percp cy5, by BD (1 mentions)
Cd16 apc h7, by BD (1 mentions)
Cd27 apc ef780, by Thermo Fisher Scientific (1 mentions)
Cd3 apc h7, by BD (1 mentions)
Cd10 pecf594, by BD (1 mentions)
Igg pe cy7, by BD (1 mentions)
Cd19 bv650, by BioLegend (1 mentions)
Cd24 bv605, by BioLegend (1 mentions)
Iga pe, by Miltenyi Biotec (1 mentions)
B cell isolation kit, by STEMCELL (1 mentions)
Cd11c apc, by BD (1 mentions)
Cd38 percp cy5, by BD (1 mentions)
Cd123 pe, by BD (1 mentions)
Foxp3 pe, by Thermo Fisher Scientific (1 mentions)
9 protocols using cd21 apc
1
Isolation and Phenotypic Analysis of Naïve B Cells
2
Comprehensive Immunophenotyping of T and B Cells
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Phenotypic analyses of T cells and B cells were performed with anti-human monoclonal antibodies (mAbs): anti-human CD3-PerCP, CD4-FITC, CD19-PerCP and CD21-APC were from BD Biosciences (San Jose, CA, USA). CXCR5-APC, ICOS-PE, PD-1-PE, IFN-γ-PE, IL-4-PE, IL-17-PE, IL-21-PE, IL-22-PE, CD27-FITC, CD86-PE, CD95-PE, CD25-APC, and Foxp3-PE antibodies were obtained from eBiosciences (San Diego, CA, USA). Cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA) and data was analyzed by FlowJo software (Tree Star, San Carlos, CA).
3
Measurement of Global DNA Methylation in PBMCs
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Global DNA methylation in PBMCs was measured as previously described [14 (link)] with a few modifications. Briefly, PBMCs (10 × 106 cells) were quickly thawed at 37°C, washed twice in RPMI-1640, resuspended in PBS (1% BSA), and incubated with CD4-PE and CD8α-Alexa Fluor 647 for 20 min at room temperature. Cells were then fixed in 2% PFA for 10 min at 37°C and permeabilised in 90% methanol for 30 min at −20°C. After three washes in PBS (1% BSA and 0.1% Tween-20), DNA was denaturated by adding 2 N HCl for 30 min (37°C) and cells were subsequently incubated in 0.1 M borate buffer for 5 min and washed three times again. PBMCs were finally resuspended in PBS (1% BSA, 0.1% Tween-20) and the following antibodies, CD3ε-PerCp5.5, CD21-APC (BD) and monocyte/granulocyte-PE, and 5-methylcytidine-DyLight488 (Novus Biologicals), were added for an incubation period of 20 min at room temperature in the dark. After two washes, cells were fixed in 1% PFA and immediately acquired on a FACSCalibur (BD).
4
Comprehensive B Cell Profiling in Blood
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B cell differentiation in peripheral blood was detected in 100 μl of blood samples washed twice with staining buffer (PBS containing 1% BSA) and stained with a cocktail of mAbs: CD45 V500, CD19 PE-Cy7, CD21 APC, IgM FITC, IgD PE, CD27 PerCP-Cy5.5, and CD38 V450 (all from BD Bioscience) for 20 minutes in the dark at room temperature. Erythrocytes were lysed with BD FACS Lysing solution (BD Bioscience) and washed twice with buffer. B cells were then classified according to their maturation stage into (i) immature (CD21-CD27-) B cells, (ii) naive (CD21+CD27-IgM+) B cells, (iii) memory B cells (CD27+CD19+), (iv) unswitched memory B cells (CD27+IgM+IgD+), (v) memory-switched B cells (CD27+IgM-IgD-), (vi) transitional B cells (CD38+IgM+), and (vii) plasmablasts (CD38+IgM-) (23 (link)) For gating strategy see Supplementary Figure S3 Table S1
5
DNA Methylation in PBMCs
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Global DNA methylation was measured in PBMCs, using surface markers (CD3-PE, CD4, or CD21-APC, CD8, or CD14-PerCP, BD Biosciences) and anti-5-methylcytidine antibody (AbD Serotec, Bio-Rad) conjugated to AF488 (Zenon AF488 Mouse IgG1 labeling kit, Life Technologies, Australia) as previously described [28 (link)].
6
Multiparametric Flow Cytometry of PBMCs
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PBMCs (5 × 106 cells) were quickly thawed at 37°C, washed once in RPMI-1640, and resuspended in PBS (1% BSA). They were then incubated with the following antibodies: CD3ε-PerCp5.5, CD4-PE, and CD8α-Alexa Fluor 647 or CD3ε-PerCp5.5, NKp46-PE, CD21-APC, and monocyte/granulocyte marker-PE (BD), for 20 min at room temperature. Cells were then washed twice with PBS (1% BSA) and fixed in 300 μL PBS 1% PFA for immediate analysis using a FACSCalibur flow cytometer (BD). Lymphocytes and monocytes were defined based on size and granularity as well as the monocytes/granulocytes marker. T-cells, B-cells, and NK-cells were defined as described above, except for the omission of CD203A staining.
7
Multiparametric B and T Cell Analysis
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The following antibodies comprising the B cell antibody panel were used: B220-V500, CD19-PerCP Cy5.5, CD23-PE, CD21-APC, CD24-PECy7, CD86-V450, MHCII-FITC, and IgM-Biotin (BD Bioscience, Erembodegem, Belgium). T cells panel consisted of the following antibodies: CD4-PerCP, CD3-AlexaFluor 700, CD62L-V500, CD44-FITC, CD28-PE, and CXCR5-V450 (BD Bioscience, Erembodegem, Belgium). Cells were acquired on a FACS Fortessa machine (BD Immunocytometry system, San Jose, CA, USA) and data was analyzed using Flowjo software (Treestar, Ashland, OR, USA).
8
Multiparameter Flow Cytometry Assay
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The following anti-human Abs were used in this study: 7-AAD PerCP, CD8 PE, CD16 APC-Cy7, CD21 APC, CD38 PE, CD56 PE-Cy7, DNAM-1 FITC, GM-CSF PerCP/Cy5.5, IFN-γ APC, Ki 67 FITC (all BD Biosciences); CD3 Pacific Blue (Life Technologies); CD4 APC, CD19 PE, (eBioscience); CD20 PE-Cy5, CD27 APC-Cy7, IgD PE-Cy7, NKp44 APC (BioLegend); CD158a, h PE-Cy5.5, CD158b1/b2, j PE-Cy5.5, CD158a, h APC, CD158b1/b2, j APC, CD159a (NKG2A) PE, CD159a (NKG2A) APC (Beckmann Coulter); hIL-12 Rß PerCP (R&D Systems). Live cells were distinguished using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Following endotoxin free recombinant human (rh) cytokines were used: rhIL-12 (R&D Systems), rhIL-2 (PeproTech), and rhIL-15 (Sigma).
9
Multicolor Flow Cytometry of PBMC
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Cryopreserved PBMC were thawed and processed if they had at least 85% viability. They were assessed by 6-color flow cytometry on the same day (FACSCanto, BD Biosciences, Franklin Lakes, USA) after cell staining and they were analyzed using the FlowJo software (TreeStar, San Jose, USA).
The following monoclonal antibodies were used to assess T, B and NK cells: CD3-FITC, CD4-PerCP, CD10-PE-Cy7, CD19-PerCP, CD38-FITC, CD38-PE-Cy7 (BD Biosciences, San Jose, USA); CD3-APC-Cy7, CD4-FITC, CD16-PE-Cy7, CD21-APC, CD27-PE, CD45-APC-Cy7, CD56-PE-Cy7, HLA-DR-PerCP, PD-1-PE (BD, San Jose, USA); 2B4-APC and FcRL4-APC (BioLegend, San Diego, USA). Isotypic controls (IgG1-FITC, IgG1-PE, IgG2a-PerCP, IgG1-PE-Cy7, IgG1-APC, IgG2b-APC, from BD Biosciences, San Jose, USA; IgG1-PE and IgG1-APC, from BioLegend, San Diego, USA) were used to evaluate nonspecific staining.
CD4+T cells were defined as CD3+CD4+ and CD8+T cells were defined as CD3+CD4-, and evaluated for frequencies of activated (CD38+HLA-DR+); or exhausted (PD-1+ or 2B4+) cell subset. B cells were defined as CD3-CD19+, and evaluated for frequencies of transitional (CD10+), naïve (CD10-CD21+CD27-), resting memory (CD10-CD21+CD27+), activated mature (CD10-CD21-CD27+) and exhausted memory B cells (CD10-CD21-CD27- or PD-1+ or FcRL4+).
NK cells were defined as CD45+CD3-CD16+CD56+ and evaluated for frequencies of PD-1+ exhausted cells.
The following monoclonal antibodies were used to assess T, B and NK cells: CD3-FITC, CD4-PerCP, CD10-PE-Cy7, CD19-PerCP, CD38-FITC, CD38-PE-Cy7 (BD Biosciences, San Jose, USA); CD3-APC-Cy7, CD4-FITC, CD16-PE-Cy7, CD21-APC, CD27-PE, CD45-APC-Cy7, CD56-PE-Cy7, HLA-DR-PerCP, PD-1-PE (BD, San Jose, USA); 2B4-APC and FcRL4-APC (BioLegend, San Diego, USA). Isotypic controls (IgG1-FITC, IgG1-PE, IgG2a-PerCP, IgG1-PE-Cy7, IgG1-APC, IgG2b-APC, from BD Biosciences, San Jose, USA; IgG1-PE and IgG1-APC, from BioLegend, San Diego, USA) were used to evaluate nonspecific staining.
CD4+T cells were defined as CD3+CD4+ and CD8+T cells were defined as CD3+CD4-, and evaluated for frequencies of activated (CD38+HLA-DR+); or exhausted (PD-1+ or 2B4+) cell subset. B cells were defined as CD3-CD19+, and evaluated for frequencies of transitional (CD10+), naïve (CD10-CD21+CD27-), resting memory (CD10-CD21+CD27+), activated mature (CD10-CD21-CD27+) and exhausted memory B cells (CD10-CD21-CD27- or PD-1+ or FcRL4+).
NK cells were defined as CD45+CD3-CD16+CD56+ and evaluated for frequencies of PD-1+ exhausted cells.
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