Fasting venous blood samples were collected from individual subjects, and peripheral blood mononuclear cells were isolated by density‐gradient centrifugation using Ficoll‐Paque Plus (Amersham Biosciences, Little Chalfont, UK). Peripheral blood mononuclear cells at 1 × 106/tube were stained in duplicate with APC‐H7‐anti‐CD3, PerCP‐Cy5.5‐anti‐CD19, PE‐Cy7‐anti‐CD27, APC‐anti‐CD38, PE‐anti‐CD138, PE‐CF594‐anti‐CD20, FITC‐anti‐IgD (BD Biosciences, San Jose, CA, USA) in the dark at room temperature for 30 min. After being washed, the cells were fixed and permeabilized using a fixation/permeabilization kit (BD Biosciences), followed by intracellular staining with BV510‐anti‐IgG, and BV421‐anti‐IgM (BD Biosciences). Negative controls were stained with isotype‐matched control antibodies (APC‐H7‐anti‐IgG1, PerCP‐Cy5.5‐anti‐IgG1, PE‐Cy7‐anti‐IgG1, APC‐anti‐IgG1, PE‐anti‐IgG1, PE‐CF594‐anti‐IgG2b, FITC‐anti‐IgG2a, BV421‐anti‐IgG1, BV510‐anti‐IgG1; BD Biosciences). The percentages of different subsets of B cells were characterized on a FACSAria II (Becton Dickinson, San Jose, CA, USA) and at least 50,000 events were analysed by FlowJo software (v5.7.2; TreeStar Inc., Ashland, OR, USA). The number of each type of cells tested was calculated, according to the percentages of this type of cells multiplied lymphocyte counts.
Anti igd fitc
Manufactured by BD
Sourced in United States
Anti-IgD-FITC is a fluorescently labeled antibody that binds to the IgD protein found on the surface of certain cells. It is used in flow cytometry applications to detect and quantify cells expressing IgD.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd27 pe, by BD (4 mentions)
Anti cd19 apc, by BD (3 mentions)
Pna biotin, by Vector Laboratories (3 mentions)
Anti igm apc, by BD (3 mentions)
Pe anti cd138, by BD (2 mentions)
Anti igm fitc, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Anti cd19 apc cy7, by BD (2 mentions)
Anti cd21 pe cy5, by BD (2 mentions)
Anti cd27 qdot 605, by Thermo Fisher Scientific (2 mentions)
Anti cd3 fitc, by BD (2 mentions)
Anti cd38 pe, by BD (2 mentions)
Anti cd73 alexa fluor 647, by BD (2 mentions)
Anti cd138 apc r700, by BD (2 mentions)
Hrp rat anti mouse igm, by BD (2 mentions)
Anti igm per cp cy5, by BD (2 mentions)
Anti b220 pe cy7, by BD (2 mentions)
Cd23 bv421, by BD (2 mentions)
Anti cd4 pe, by Thermo Fisher Scientific (2 mentions)
Anti b220 apc, by Thermo Fisher Scientific (2 mentions)
22 protocols using anti igd fitc
1
Quantification of B Cell Subsets
2
Multiparametric Flow Cytometry Analysis
3
Multiparametric Flow Cytometry of Splenic B Cells
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The surface expression level of CD23 on splenic B cells was analyzed by flow cytometry. B cells were pre-incubated with anti-CD16/CD32 antibody (BD Biosciences, San Jose, CA, USA, Cat. No 553142) to block FcγR, and following by PerCP-Cy5.5-anti-B220 (BD Biosciences, Cat. No 552771), PE-anti-CD138 (BD Biosciences, Cat. No 553714), PE-Cy7-anti-IgM (BD Biosciences, Cat. No 552867), biotin-anti-IgD (Southern Biotech, Birmingham, AL, USA, Cat. No 1120-08), FITC-anti-IgD (BD Biosciences, Cat. No 553439), FITC-anti-IgM (BD Biosciences, Cat. No 553408) and APC-NP19, and FITC-anti-CD23 antibodies (BD Biosciences, Cat. No 553138), plus AF405- streptavidin (Thermo Scientfic, Waltham, MA, USA ,Cat. No S32351). After washing and fixation, cells were analyzed using a flow cytometer (BD CantoII, BD Biosciences). Data were analyzed using FACSDiva (BD Biosciences) and FlowJo (Treestar Inc. Ashland, OR, USA) software.
4
Immunofluorescent Staining of Splenic Tissue
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Splenic tissue was sectioned and stained as previously described [14 (link)]. Briefly, spleens were snap-frozen in OCT compound (Sakura Finetek, Torrance, CA) at the time of sacrifice. Cryostat spleen sections (5 μm) were fixed in acetone, washed with PBS, and blocked with 5% fetal bovine serum in PBS prior to staining with FITC-anti-IgD (BD Biosciences) or -anti-IgMa (BD Biosciences), biotin-conjugated anti-PNA (Sigma-Aldrich), and PE-anti-IgMa (BD Biosciences) or -anti-CD4 (BD Biosciences). Biotinylated antibody staining was revealed with 7-amino-4-methylcoumarin-3-acetic acid-conjugated streptavidin (Jackson ImmunoResearch) as a secondary reagent. Sections were mounted with Fluoro-Gel (Electron Microscopy Sciences) and fluorescence was visualized after 24–48 hr using a Zeiss Axioplan 2 imaging microscope (Zeiss, Oberkochen, Germany). Images were processed using ImageJ software (ImageJ, National Institutes of Health, Bethesda, Maryland).
5
Multiparameter Flow Cytometry of PBMCs
6
Comprehensive Immunological Protocols for Flow Cytometry, Histology, and ELISA
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All chemicals were purchased from Sigma–Aldrich unless otherwise stated.
For flow cytometry we used: washing buffer, PBS containing 0.1% NaN3; staining buffer, PBS containing 0.1% BSA, and 0.1% NaN3; fixation buffer: PBS containing 0.1% paraformaldehyde.
For histological staining, we used: washing buffer, PBS; blocking buffer, PBS containing 5% BSA.
For ELISA we used: blocking buffer, PBS containing 1.5% non-fat dry milk; washing buffer, PBS containing 0.05% or 0.5% Tween-20 was used as for cytokine- or antibody ELISA.
The following monoclonal antibodies were used for flow cytometry (also seeSupplementary Table S1 ): anti-CD3-FITC, anti-CD4-PE, anti-CD8a-PE-Cy5, anti-CD25-APC, anti-CD38-PE, anti-CD73-Alexa Fluor 647, anti-IgD-FITC, anti-IgM-PerCP-Cy5.5, anti-CD138-APC-R700, anti-B220-PE-Cy7, CD23-BV421, all from BD Bioscience (San Jose, CA, USA). For immunofluorescence: anti-B220-Alexa Fluor 647 and anti-CD3-FITC. For immunohistochemistry: rat anti-mouse IgD (BD Bioscience) and PNA-Biotin (Vector Laboratories, BioMarker, Gödöllő, Hungary). Extravidin-alkaline phosphatase (Sigma-Aldrich) or ImmPRESS goat anti-rat IgG-HRP polymeric conjugate (Vector Laboratories, BioMarker, Gödöllő, Hungary) was used to detect the PNA, or IgD antibody, respectively. For ELISA: HRP rat anti-mouse-IgG1, HRP rat anti-mouse IgG2a and HRP rat anti-mouse-IgM (BD Bioscience) were used.
For flow cytometry we used: washing buffer, PBS containing 0.1% NaN3; staining buffer, PBS containing 0.1% BSA, and 0.1% NaN3; fixation buffer: PBS containing 0.1% paraformaldehyde.
For histological staining, we used: washing buffer, PBS; blocking buffer, PBS containing 5% BSA.
For ELISA we used: blocking buffer, PBS containing 1.5% non-fat dry milk; washing buffer, PBS containing 0.05% or 0.5% Tween-20 was used as for cytokine- or antibody ELISA.
The following monoclonal antibodies were used for flow cytometry (also see
7
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).
8
Isolation and Culture of Naïve B Cells
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After PBMC isolation, B cells were enriched by positive selection using magnetic columns according to the manufacturer’s instructions (EasySepTM, StemCell Technology, Vancouver, BC, Canada). The cells were stained with anti-CD19-PerCP-Cy5.5, anti-CD27-PE, anti-IgM-APC, and anti-IgD-FITC (all antibodies from BD Biosciences, San Diego, CA, USA). The B cells, defined as CD19+CD27−IgD+IgM−, were sorted with FACSAria II cell sorter (Beckton-Dickinson, San Jose, CA, USA). After sorting, the cells were labeled with 5 µM of CFSE dye (Invitrogen, Eugene, OR, USA) before the culture, according to manufacturer’s instructions. The cells were cultured in AIM-V serum-free medium in the presence of CpG-ODN2006 (InvivoGen, San Diego, CA, USA) (2.5 µg/mL) and anti-CD40 (BD Bioscience, San Diego, CA, USA) (2 µg/mL) in a 96-well U bottom plate at 37 °C in a humidified 5% CO2 incubator. On day 5, the cells were harvested and acquired using FACSCelesta (Beckton-Dickinson, San Jose, CA, USA).
9
Quantification of Germinal Center Size
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Unfixed spleens were frozen in O.C.T. compound, and 10-µm-thick sections were cut with a Microm HM 550 cryostat (Thermo Fisher Scientific). Sections were fixed in acetone at −20°C for 5 min and stained with anti–GL7-PE and anti–IgD-FITC (BD). Fluorescent images were scanned with a NanoZoomer 2.0-HT equipped with a Fluorescence Imaging Module (Hamamatsu Photonics). Germinal center areas were quantified using NDP.view software (Hamamatsu Photonics). Distinct GL7+ germinal center and surrounding IgD+ follicle perimeters were manually defined, and areas were calculated. Three Zbtb20+/+ and Zbtb20trap/trap chimeras each were analyzed. Six sections of ∼10-mm2 area, spaced at least 120 µm apart from each other in the z-dimension, were analyzed for each animal. For presentation, contrast adjustment was applied equally to the representative images of both genotypes shown in Fig. 4 B using Photoshop CS3 (Adobe).
10
Multiparametric Flow Cytometry of Immune Cells
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The distribution of the different leukocyte subsets was characterized by multiparametric flow cytometry 0–10 days before the first vaccine dose, following the Guidelines for the use of flow cytometry and cell sorting in immunological studies. To this end, 200 µl of whole fresh blood were stained with anti-CD45 Pacific Orange, anti-CD3 APC, anti-CD4 FITC, anti-CD8 APC H7, anti-CD19 PerCP, anti-CD27 PE, anti IgD FITC and anti-IgM APC (BD Becton Dickinson). Lymphocyte populations were identified as follows: CD4+ lymphocytes: CD45+ CD3+, CD4+, CD8-; naïve B cells: CD45+ CD3- CD19+ CD27- IgM+ IgD+. Absolute cell numbers were calculated from white blood cell counts obtained with an XN-10 Hematology System (Sysmex, Kobe, Japan- Roche, Basel, Switzerland).
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