PBMCs were isolated from peripheral blood samples collected on days 0 (n = 9), 7 (n = 17), and 28 (n = 17) using Lymphoprep (Abbott Diagnostics, Lake Forest, IL, USA), and were stained with CD2 (clone RPA‐2.10; BioLegend, San Diego, CA, USA), CD3 (clones HIT3a and UCHT1; BioLegend), CD4 (clone RPA‐T4; BioLegend), CD10 (clone eBioCB‐CALLA; Thermo Fisher Scientific), CD19 (clone HIB19; BioLegend), CD20 (clone 2H7; BioLegend), CD27 (clone O323; Thermo Fisher Scientific), CD38 (clone HIT2; Thermo Fisher Scientific), and IgD (clone IA6‐2; BD Biosciences, San Jose, CA, USA) in the presence of human Fc receptor (FcR) block (Miltenyi Biotec, Bergisch Gladbach, Germany). CD19+ CD38++ CD27++ cells that were negative for CD2, CD3, CD4, CD10, IgD, and CD20 were counted as plasma cells.14 Data were acquired on an FACS Canto II (BD Biosciences) and analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
Igd clone ia6 2
Manufactured by BD
Sourced in United States
IgD (clone IA6-2) is a monoclonal antibody that binds to the IgD molecule. It is used in research applications to detect and analyze IgD-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Lymphoprep, by Abbott (1 mentions)
Cd20 clone 2h7, by BioLegend (1 mentions)
Cd4 clone rpa t4, by BioLegend (1 mentions)
Cd2 clone rpa 2.10, by BioLegend (1 mentions)
Cd19 clone hib19, by BioLegend (1 mentions)
Fcr block, by Miltenyi Biotec (1 mentions)
Lsr 2 cytometer, by BD (1 mentions)
Facsaria 2 sorp cell sorter, by BD (1 mentions)
Cd3 clone sp34 2, by BD (1 mentions)
Fixable viability dye eflour 506, by Thermo Fisher Scientific (1 mentions)
2 protocols using igd clone ia6 2
1
PBMC Isolation and Flow Cytometry Analysis
2
Multiparametric Flow Cytometry of Naive B Cells
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As Kaminski et al. (24 (link)) described, multiparametric Flow Cytometry was used. Cells were characterized using CD3 (Clone SP34-2, BD Biosciences NJ, United States), CD19 (Clone SJ25C1 BD Biosciences), CD38 (Clone HIT2, Ebioscience, San Diego, CA, United States), CD27 (Clone L128, BD Biosciences), IgD (clone IA6-2, BD Biosciences), CD11c (Clone B-ly6, BD Bioscience), CD21 (Clone B-ly4, BD Bioscience), and CXCR5 (Clone 51505, R&D) markers. Also, we excluded CD38++ CD27++ Plasma Cells and dead cells with a Fixable Viability Dye eFlour 506 (Thermo Fisher, Scientific, Waltham, MA, United States). Anti-VH4.34 antibody (clone 9G4) was also used. The markers IgD+ CD27− CXCR5+ CD11c− were used for sorting resting Naive B cells. For compensation, we used beads stained with each fluorochrome. A BD LSR II cytometer was used for reading and BD FACS Aria II SORP Cell Sorter. The B cell binding index (BCB) was calculated as described in Jenks et al. (25 ), using the following formula [Resting naive 9G4 + median fluorescence intensity]/[SWM 9G4 + median fluorescence intensity]. FlowJo™ v9/10 Software (BD Life Sciences) was used for cytometric analysis.
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