Mononuclear cells from bone marrow and spleen were prepared as previously reported (4 (link), 18 ). B cell sub populations were separated based on expression of specific surface proteins. Early B cell subsets in the bone marrow were identified using the scheme of Melchers (19 (link)). Transitional B cell subsets were identified using the scheme of Allman (20 (link)). Marginal zone and follicular B cells were identified using the scheme of Loder (21 (link)). The following sets of monoclonal antibodies were used: Anti-B220 (PerCP) [BD Pharmingen, San Diego, CA], anti-BP-1 (PE) (a gift from John Kearney, UAB), and anti-IgM (Cy5) [Jackson ImmunoResearch], West Grove, PA], anti-cKit (allophycocyanin) [BD Pharmingen, San Diego, CA], CD19 (SPRD) [Southern Biotech, Birmingham, AL], anti IgD (biotin) [BD Pharmingen], anti CD21 (PE) [BD Pharmingen], anti-CD25 (FITC) [BD Pharmingen, San Diego, CA], anti CD21 (PE) [BD Pharmingen, San Diego, CA], anti AA4.1 (Pecy7) [ebioscience, San Diego, CA] and anti CD23 (FITC) [BD Pharmingen, San Diego, CA].
Anti cd21 pe
Manufactured by BD
Sourced in United States
The Anti-CD21-PE is a fluorochrome-conjugated monoclonal antibody that binds to the CD21 surface antigen. CD21 is a complement receptor expressed on mature B cells and a subset of T cells. This antibody can be used for the identification and enumeration of B cells in flow cytometric analysis.
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Lab products found in correlation
Anti cd27 apc, by BD (2 mentions)
Anti aa4.1 pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti c kit, by BD (1 mentions)
Anti cd23 fitc, by BD (1 mentions)
Anti cd25 fitc, by BD (1 mentions)
Anti b220 percp, by BD (1 mentions)
Facs lysis solution, by BD (1 mentions)
Fcs express software, by De Novo Software (1 mentions)
Anti hla dr percp, by BD (1 mentions)
Anti cd20percp, by BD (1 mentions)
Anti cd3 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd34 fitc, by BD (1 mentions)
Anti cd11c pe, by BD (1 mentions)
Anti cd45 apc, by BioLegend (1 mentions)
Pe anti igg1, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Anti igg1 fitc, by BD (1 mentions)
Anti cd20 percp cy5, by BD (1 mentions)
Apc mouse igg1 k isotype control, by BD (1 mentions)
Anti cd19 bv421, by BD (1 mentions)
5 protocols using anti cd21 pe
1
Isolation and Identification of B Cell Subsets
2
Multicolor Flow Cytometry of Immune Cells
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Direct immunofluorescence with specific antibodies was performed either on peripheral blood as previously described with some modifications using the following antibodies: anti-IgG1 FITC, anti-CD34-FITC, anti-IgG1-PE, anti-CD3-FITC/CD16+CD56-PE, anti-CD11C-PE, anti-CD21-PE, anti-CD20-PerCP and anti-HLA-DR-PerCP (BD Bioscience, San Jose, CA), anti-CD19-FITC, anti-CD3-APC (Invitrogen, Carlsbad, CA, anti-Lineage-FITC (anti CD3/CD14/CD16/CD19/CD20/CD56), anti-CD45-APC (Biolegend, San Diego, CA, USA) and anti-BDCA2-APC (Miltenyi Biotech, San Diego, CA, USA) [13 (link),21 (link)]. Briefly, after washing the whole blood with PBS, 100ul of blood was stained with the relevant cocktail of antibodies at 4 °C for 30 min followed by red blood cell lysis using BD FACS lysis solution (BD Bioscience, San Jose, CA, USA). Samples were acquired on Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FCS express software (De Novo software, Glendale, CA, USA). During acquisition, a lymphocyte gate was assigned, and 10,000 events were collected for the T cells and NK cells. For DCs, a million ungated events were acquired.
3
Phenotypic Characterization of B-Cell Subsets
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A total of 0.5 x 106 PBMCs were stained with the following combination of antibodies: Anti-CD19-BV421 (clone HIB19), anti-CD20-PerCPCy™5.5 (clone L27), anti-CD21-PE (clone B-ly4), anti-CD27-APC (clone M-T271) and anti-CD45-V500 (clone HI30). Anti-CD19-BV421, anti-CD20-PerCPCy™5.5, anti-CD21-PE, anti-CD27-APC, anti-CD45-V500, PerCP-Cy5.5 mouse IgG1 k isotype control, BV421 mouse IgG1 k isotype control, V500 mouse IgG1 k isotype control, PE mouse IgG1 k isotype control, and APC mouse IgG1 k isotype control were purchased from BD Biosciences (Becton Dickinson, Franklin Lakes, NJ, USA). B-cell populations were identified based on the expression of the following markers: activated memory B-cells (CD19+ CD20+ CD21- CD27+ CD45+), resting memory B-cells (CD19+ CD20+ CD21+ CD27+ CD45+), tissue-like memory cells (CD19+, CD20+ CD21- CD27- CD45+), and resting naïve B-cells (CD19+ CD20+ CD21+ CD27- CD45+).
CD34+ hematopoietic progenitors in peripheral blood were enumerated using the Stem Cell Enumeration Kit (Becton Dickinson).
All flow cytometry analyses were performed using a BD LSRFortessa™ Flow Cytometer (Becton Dickinson) from the I2MC cytometry platform (CHU Rangueil, Toulouse, France) and BD FACSDiva™ Software (Becton Dickinson).
CD34+ hematopoietic progenitors in peripheral blood were enumerated using the Stem Cell Enumeration Kit (Becton Dickinson).
All flow cytometry analyses were performed using a BD LSRFortessa™ Flow Cytometer (Becton Dickinson) from the I2MC cytometry platform (CHU Rangueil, Toulouse, France) and BD FACSDiva™ Software (Becton Dickinson).
4
Phenotyping of B-cell Subsets in PBMC
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B-cell subsets were measured in freshly thawed cryopreserved PBMCs. After washing and counting viable cells, PBMCs were surface-stained with the following conjugated mAbs: anti-CD19-PE-Cy5, anti-CD21-PE, anti-CD20-PE-Cy7, anti-CD27-APC, anti-IgM-FITC, anti-CD38-FITC, anti-HLA-DR-PE-Cy7 (BD Bioscience); anti-CD10-APC-Cy7, anti-BAFFr-APC-Cy7 (Biolegend); anti-TACI-PE, anti-CxCr5-APC (R & D Systems) and analyzed with Guava easyCyte 8HT (Millipore) and FlowJo (Treestar). Subsets were expressed as percentages of the parent CD19+ B-cell population.
5
Comprehensive Immunological Profiling Protocol
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Serum immunoglobulins (Ig) levels (IgG, IgA and IgM) were measured by immunoturbidimetry using the automatic analyzer Alinity c system (Abbott Laboratories, Chicago, IL, USA). For cellular evaluation, EDTA whole blood samples were collected. Lymphocyte subpopulations (CD4+ T, CD8+ T, B and NK cells) were performed using BD Trucount tubes and the BD Multitest 6 Color BTNK kit (BD Biosciences, San Diego, CA, USA), which included the following mixtures of fluorophore-conjugated monoclonal antibodies (mAb): anti-CD45-PerCP-Cy5.5, anti-CD3-FITC, anti-CD8-APC-Cy7, anti-CD4-PE-Cy7, anti-CD19-APC, and anti-CD16+CD56-PE. B cell phenotype was performed with an eight-color panel of the following mAb: anti-CD45-APC-H7, anti-CD19-V500, anti-CD10-V450, anti-CD38-PE-Cy7, anti-CD21-PE, anti-CD27-PerCP-Cy5, anti-IgD-FITC, and anti-IgM-APC (BD Biosciences, San Diego, CA, USA), following EURO-Class classification. Cells were acquired on a BD FACSCanto II Flow Cytometer (BD Biosciences, San Diego, CA, USA), and the InfinicytTM22.0 software was employed for multiparametric analysis (Cytognos SL, Salamanca, Spain).
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