Lymphoma immunophenotype was assessed as previously described [22 (link)]. The following antibodies were used for staining: CD19-APC, B220 (CD45R)-FITC, IgM biotin, IgD-PE, and Streptavidin-APC/Cy7 (BD Biosciences, USA). Data were collected by a flow cytometer (BriCyteE6, Mindray Bio- Medical Electronics Co. Ltd., Shenzhen, China) and analyzed using FlowJo Version 10.1 software. For the analysis of tumor-associated macrophages, cell suspensions of lymph nodes were obtained by grinding and filtering tissues through 0.4-μm cell strainers (BD Biosciences, USA) in PBS. Cells were then transferred to a fresh tube for centrifugation at 1000× g for 5 min. The cell pellet was incubated in red blood cell lysis buffer (Lonza) for 1 min at room temperature, diluted in PBS, and centrifuged for 1000× g for 5 min. The cell pellet was incubated with fluorescent-conjugated antibodies (dilution 1:50 in PBS) for 15 min at 4 °C in the dark, washed, and analyzed by flow cytometry. The following antibodies were used: F4/80 (6F12) from Miltenyi; CD11b (M1/70.15.5) and Gr1 (RB6–8C5) from BD Pharmingen.
Igd pe
Manufactured by BD
Sourced in United States
The IgD PE is a fluorescent-labeled antibody used in flow cytometry applications for the detection and analysis of IgD-expressing cells. It provides a reliable tool for immunophenotyping and cell population characterization.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (4 mentions)
Cd38 pe cy7, by BD (4 mentions)
Cd19 pe cy7, by BD (3 mentions)
Cd138 apc, by BD (3 mentions)
Cd20 percp cy5, by BD (3 mentions)
Cd27 apc, by BD (3 mentions)
Cd27 apc efluor780, by Thermo Fisher Scientific (3 mentions)
Cd19 apc, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Cd3 fitc, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Cd45 v500, by BD (2 mentions)
Cd23 pe, by BD (2 mentions)
Cd19 percp cy5, by BD (2 mentions)
Facscanto 2 flow cytometer, by FlowJo (2 mentions)
Cd20 apc h7, by BD (2 mentions)
Lsrii cytometer, by BD (2 mentions)
Cd19 pc5, by Beckman Coulter (2 mentions)
Cd27 fitc, by Agilent Technologies (2 mentions)
Igm apc, by Jackson ImmunoResearch (2 mentions)
18 protocols using igd pe
1
Immunophenotyping of Lymphoma and TAMs
2
Multiparametric Flow Cytometric Immunophenotyping
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Peripheral blood mononuclear cells were washed in PBS with 1% BSA (bovine serum albumin) and incubated for 30 min at 25°C with fluorescently labeled antibodies specific for B and T cells. Subsequently, samples were centrifuged and resuspended in propidium iodide (PI) solution (1 μg/ml PI and 10 μg/ml RNase A in PBS) and analyzed using BD FACS Canto II flow cytometer (BD Biosciences). The B cell panel included the following antibodies: IgA FITC, IgD PE, CD3 PerCP-Cy5.5, CD27 APC-H7, CD19 PE-Cy7, β7 APC, and CD38 BV421 from BD Biosciences. The T cell panel included the following antibodies: CXCR5 BV421, CXCR3 PE, CD4 APC-H7, CD3 FITC, CD196 APC, CD279 (PD-1) BV510, and CD45RA PE-Cy7 from BD Biosciences. Results were analyzed using FACSDiva software (BD Biosciences) and reported as MFI, reflecting the levels of cell surface antigens and relative cell count with respect to hierarchically higher cell populations (%). Cellular aggregates were eliminated using morphology parameters (FSC-A and FSC-H) (see Supplementary Figures 1 , 2 for gating strategy).
3
Phenotypic Analysis of Cell Suspensions
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Phenotypic analysis of cell suspensions was performed by flow cytometry. Cells were stained with a panel of seven 8-colour antibody combinations (Table 1 ). The following 6 monoclonal antibodies were used as a “backbone” in all combinations: CD38-PerCP5.5, CD10-APC, CD19-APC-H7, CD5-V450, CD45-V500 (BD Biosciences) and CD27-PeCy7 (Beckman Coulter). All FITC- or PE-labelled antibodies were specific of a particular combination, i.e. CD20-FITC, CD44-FITC, CD24-PE, CD40-PE, (Beckman Coulter); CD43-FITC, CD81-FITC, CD86-FITC, CD21-PE, CD22-PE, CD23-PE, CD268 (BAFF-R)-PE, IgD-PE, (BD Biosciences) IgM-FITC (Dako). Clone and isotype specificity of these antibodies are detailed in S1 Table ; the antibodies were used at the dilution recommended by the manufacturers.
Samples containing 106 cells in a volume of 100 μl were incubated with the combination of antibodies at the supplier recommended concentration during 20 minutes in the dark, at 4°C. After erythrocytes lysis with NH4Cl at 4°C for 5 minutes, samples were washed with HBSS medium (Gibco).
Analysis was performed using 3-laser, 8-colour BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software version 6 (BD Biosciences). At least 104 cells were acquired in the CD19+ gate. BD CompBeads (BD Biosciences) were used for compensation settings. Cytometer performances were checked daily using CST beads (BD Biosciences).
Samples containing 106 cells in a volume of 100 μl were incubated with the combination of antibodies at the supplier recommended concentration during 20 minutes in the dark, at 4°C. After erythrocytes lysis with NH4Cl at 4°C for 5 minutes, samples were washed with HBSS medium (Gibco).
Analysis was performed using 3-laser, 8-colour BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software version 6 (BD Biosciences). At least 104 cells were acquired in the CD19+ gate. BD CompBeads (BD Biosciences) were used for compensation settings. Cytometer performances were checked daily using CST beads (BD Biosciences).
4
Multiparametric Phenotyping of B Cells
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PBMCs were resuspended in phosphate-buffered saline (PBS), containing 0.5 % (w/v) BSA and 0.01 % sodium azide. PBMCs were incubated with saturating concentrations of fluorescently labeled conjugated monoclonal antibodies (MoAbs). Analysis of cells was performed using a FACSCanto-II flowcytometer and FlowJo software. The following directly conjugated MoAbs were used for flow cytometry: CD19-PerCP-Cy 5.5, CD19 Alexa-700, CD20-APC, CD20-PerCP-Cy 5.5, CD38 PE-Cy7, CD138 APC, IgG-PE, IgD-PE, and IgA-PE from BD-biosciences (San Jose, USA), CD27-FITC from Sanquin (Amsterdam, the Netherlands), IgM-PE from ITK-diagnostics (Uithoorn, the Netherlands).
5
Phenotypic Analysis of Cryopreserved PBMCs
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Cryopreserved PBMCs were thawed and washed twice with 10 mL of FACS buffer (1 x PBS containing 2% FBS and 1 mM EDTA) and resuspended in 100 uL of 1x PBS containing Zombie UV live/dead dye at 1:200 dilution (BioLegend, 423108) and incubate at room temperature for 15 minutes. Following washing, cells were incubated with an antibody cocktail for 1 hour protected from light on ice. The following antibodies were used: IgD PE (BD Biosciences, 555779), IgM PerCP-Cy5.5 (BioLegend, 314512), CD20 APC-H7 (BD Biosciences, 560734), CD27 PE-Cy7 (BioLegend, 302838), CD14 PE/Dazzle™ 594 (BioLegend, 301852), CD16 BV605 (BioLegend, 302040), IgG BV650 (BD Biosciences, 740596), CD3 BUV737 (BD Biosciences, 612750) and Alexa Fluor 488-labeled Wuhan spike (SinoBiological, 40589-V27B-B), and BV421 labeled Omicron Spike (SinoBiological™, 40589-V49H3-B). All antibodies were used as the manufacturer’s instruction and the final concentration of each probe was 0.1 ug/ml. Cells were washed twice in FACS buffer and immediately acquired on a BD FACS Aria III for acquisition and FlowJo for analysis.
6
Four-color Flow Cytometry of Memory B Cells
7
Flow Cytometry Analysis of Eμ-Myc Lymphoma
8
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
9
Characterization of B-cell and T-cell subsets
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In separate experiments, CFSE-labeled PBMCs were resuspended in a culture medium and incubated with saturating concentrations of dye-conjugated mAbs for 30 min at 4°C. Naïve (CD20+CD19+IgD+CD27−), non-switched (CD20+CD19+IgD+CD27+), and memory (CD20+CD19+IgD−CD27+) B-cell populations and non–B-cell populations (CD19−) were isolated by FACS with a FACSAria II (BD Biosciences) dependent on the experiment. In addition, from the non–B-cell fraction, total CD3+ T cells (CD3+CD19−) were isolated for specific experiments. The following mAbs were used for isolation: CD3 PE (347247; BD Biosciences), CD19 APC-R700 (564977; BD Biosciences), CD27 APC (337169; BD Biosciences), and IgD PE (555779; BD Biosciences). Different combinations, at a fixed number of cells (25,000 B cells with 125,000 non-B cells) or 100,000 T cells, were then cultured in 96-well U-bottom plates for 6 d. During culture, the cells were stimulated with CpG or αCD40 + IL-21 with or without concentrations of daratumumab as described above. T cells were stimulated with anti-CD3 (αCD3) (clone 1xE; Sanquin) and 10 μg/ml anti-CD28 (αCD28) (clone 15E8; Sanquin) with or without daratumumab. After 6 d, cells were analyzed by flow cytometry as described above. Supernatants were collected for further analysis.
10
Multicolor Flow Cytometry of B-cells
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Four-color flow cytometry with LSRII cytometer and FacsDIVA software (BD Biosciences) was performed on patient PBMCs using anti–human mAbs CD19 PC5 (Beckman Coulter, IM2643U), CD27 FITC (Dako, F7178), IgM APC (Jackson ImmunoResearch, 309605095), and IgD PE (BD Pharmingen, 555779). Analysis was performed with FlowJo software.
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