Freshly prepared PBMCs were used. Subpopulations of T cells, B cells, natural killer (NK) cells and antigen-presenting cells (APC) were characterized by surface staining with fluorescence labelled anti-CD3, anti-CD4, anti-CD5, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD27, anti-CD38, anti-CD45RA, anti-CD45RO, anti-CD56 and anti-HLA-DR (BD Bioscience); anti-BDCA1, anti-BDCA2, anti-BDCA3, anti-BDCA4 and anti-slan (Miltenyi Biotec). Negative controls included directly labeled or unlabeled isotype-matched irrelevant antibodies (BD Biosciences). Freshly prepared CSF cells were used directly for FACS analysis. Fluorescence-labeled antibodies for surface staining were used as follows: anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD19, anti-CD25, anti-CD27, anti-CD45RA, anti-CD45RO, anti-CD56 and anti-HLA-DR (BD Bioscience); anti-BDCA1 and anti-slan (Miltenyi Biotec). All cells were measured on a LSR-Fortessa (BD Biosciences) and evaluated by FACS-Diva Software (BD Bioscience).
Anti cd27
Manufactured by BD
Sourced in United States
Anti-CD27 is a monoclonal antibody that binds to the CD27 receptor. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on the surface of various immune cells. The primary function of Anti-CD27 is to specifically target and detect the CD27 receptor for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd19, by BD (22 mentions)
Anti igd, by BD (14 mentions)
Lsrfortessa, by BD (10 mentions)
Anti cd3, by BD (10 mentions)
Anti cd38, by BD (8 mentions)
Anti cd45, by BioLegend (7 mentions)
Anti cd8, by BD (5 mentions)
Facsdiva software, by BD (4 mentions)
Anti cd4, by BD (4 mentions)
Live dead detection kit, by Thermo Fisher Scientific (4 mentions)
Diva software, by BD (4 mentions)
Anti igd bv510, by BD (4 mentions)
Anti cd20, by BD (3 mentions)
Anti cd21, by BioLegend (3 mentions)
Anti cd95, by BioLegend (3 mentions)
Anti cd11c, by BioLegend (3 mentions)
Facscanto 2, by BD (3 mentions)
Facscalibur ow cytometer, by BD (3 mentions)
Anti cd38 percp cy5.5, by BD (3 mentions)
Anti cd45ra, by BD (2 mentions)
32 protocols using anti cd27
1
Comprehensive Immune Cell Profiling
2
Comprehensive PBMC Immunophenotyping Assay
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Approximately 250,000 PBMC were stained with monoclonal antibodies (Becton-Dickinson, San Diego, CA, USA): anti-CD3 [fluorescein isothiocyanate (FITC)], anti-CD8 [peridinin chlorophyll protein (PerCP)] and anti-CD38 [phycoerythrin (PE)]; and anti-CD19 [peridinin chlorophyll protein (PerCP)], anti-CD10 [phycoerythrin (PE)-TexasRed], anti-CD21 [fluorescein isothiocyanate (FITC)], anti-CD27 [phycoerythrin (PE)-Cy7], anti-CD80 [allophycocyanin-H7 (APC-H7)] and anti-CD86 [allophycocyanin (APC)]. Appropriate isotype controls (mouse IgG1-PE and mouse IgG2b-APC) were used to evaluate non-specific staining. All the samples were analyzed by LSR II cytofluorimeter (Becton-Dickinson). A total of 50,000 events was collected in the lymphocyte gate using morphological parameters (Forward and Side scatter). Data were processed with FACSDiva™ Software (Becton-Dickinson) and analysed using Kaluza® Analyzing Software v.1.2 (Beckman Coulter, Fullerton, CA, USA).
3
Isolation and Characterization of B Cell Subsets
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Peripheral blood lymphocytes (PBL) B cells were negatively selected by B cells Isolation Kit II (Miltenyi cat# 130‐091‐151). B cell subpopulations were isolated by FACS sorting (FACS ARIA II, Becton Dickinson) as reported previously20 by staining the cells with anti‐sIgD (FITC), anti‐sIgM (PE), anti‐CD27 (PECy5), and anti‐CD19 (APC‐Vio770). The sorted B cell subpopulations were identified as reported in Table S1 .
Spleen and tonsil cell suspensions were obtained using a gentle MACS dissociator (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) and mononuclear cells separated by density gradient separation Ficoll Hypaque (Seromed; Biochrom KG, Berlin, Germany).
For B‐cell subpopulations cell sorting, the following combinations of mAbs was employed: anti‐sIgD (Alexa 488; Biolegend, San Diego CA), anti‐CD19‐APC‐Vio770 (Milteni Biotech), anti‐sIgM‐PE (Dako), anti‐CD38 (PC7, BD), anti‐CD27 (PECF594, BD), anti‐CD24 (PC5, BD). Only naïve B cells, IgM memory (M‐Mem), and switched‐memory (S‐Mem) B cells were included in this study. In tonsils, memory B cells (M‐Mem) were isolated as IgD‐low/CD38−CD27+, thus comprising IgM+ and switched memory B cells. Moreover, germinal center (GC) B cells were isolated as IgD−/CD38+/CD24− B cells.
Spleen and tonsil cell suspensions were obtained using a gentle MACS dissociator (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) and mononuclear cells separated by density gradient separation Ficoll Hypaque (Seromed; Biochrom KG, Berlin, Germany).
For B‐cell subpopulations cell sorting, the following combinations of mAbs was employed: anti‐sIgD (Alexa 488; Biolegend, San Diego CA), anti‐CD19‐APC‐Vio770 (Milteni Biotech), anti‐sIgM‐PE (Dako), anti‐CD38 (PC7, BD), anti‐CD27 (PECF594, BD), anti‐CD24 (PC5, BD). Only naïve B cells, IgM memory (M‐Mem), and switched‐memory (S‐Mem) B cells were included in this study. In tonsils, memory B cells (M‐Mem) were isolated as IgD‐low/CD38−CD27+, thus comprising IgM+ and switched memory B cells. Moreover, germinal center (GC) B cells were isolated as IgD−/CD38+/CD24− B cells.
4
Immunophenotyping of B-cell Subsets
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FITC, PE or PerCP labeled anti-CD19, anti-CD27, anti-IgD, anti-BAFF-R, and anti-TACI monoclonal antibodies (Becton Dickinson, San José, CA, USA) were used. The cells were evaluated using a FACScan flow cytometer and FCS 3 Express, De Novo Software (Los Angeles, CA, USA).
5
Comprehensive Immunophenotyping of Cryopreserved PBMC
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Immunophenotyping was performed on cryopreserved PBMC. Cells were thawed, washed, stained for 20 min in the dark with the Live/Dead Fixable Near-IR Dead Cell Stain Kit (Life Technologies, Carlsbad, California, USA) and the following labelled monoclonal antibodies (mAbs): anti-CD3 [fluorescein isothiocyanate (FITC)], anti-CD4 [peridinin chlorophyll protein (PerCP)], anti-CD38 [phycoerythrin (PE)], anti-HLA-DR [allophycocyanin (APC)], anti-CD279 (programmed cell death 1, PD-1) [PE-Cy7], anti-CD57 [PE], anti-CD21 [BV421], anti-CD27 [PE-Cy7], anti-IgD [PE], anti-CD274 (programmed cell death ligand 1, PD-L1) [BV421] (Becton-Dickinson, San Diego, California, USA); anti-CD8 [VioGreen], anti-CD28 [APC], anti-CD19 [VioBright515], anti-CD10 [APC] (Miltenyi Biotec, Auburn, California USA). Cells were then washed and resuspended in phosphate-buffered saline supplemented with 1% paraformaldehyde. All samples were analyzed using LSRII Flow cytometer (Becton-Dickinson). A total of 50000 events were collected in the lymphocyte gate using morphological parameters (forward and side-scatter). Data were processed with FACSDiva Software (Becton-Dickinson) and analyzed using Kaluza Analyzing Software v.1.2 (Beckman Coulter) ( Figure 1 supplementary ).
6
Phenotyping B Cell Subsets
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After thawing, PBMC (2 × 106/ml) were stained for 20 min at room temperature with the following antibodies: anti-CD45 (Biolegend 368540), anti-CD19 (BD 555415), anti-CD27 (BD 555441) and anti-IgD (BD 555778) to measure naive (IgD + CD27-), IgM memory (IgD + CD27+), switched memory (IgD-CD27+), and DN (IgD-CD27-) B cells. Up to 104 events in the B cell gate were acquired on an LSR-Fortessa (BD) and analyzed using FlowJo 10.0.6 software. Single color controls were included in every experiment for compensation. Isotype controls were also used in every experiment to set up the gates.
7
B Cell Subset Profiling and Analysis
8
Isolation and Characterization of B Cells from Peripheral Blood
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PB samples were obtained after written informed consent was received from participants prior to inclusion in the study according to the Declaration of Helsinki, and approval by the ethics committee of the Medical Faculty at the University of Duisburg‐Essen, Germany (BO‐10‐4380). B cells were isolated by Ficoll density centrifugation (density 1.077 g/ml, Pan BioTech, Aidenbach, Germany) followed by staining with anti‐CD3 (BD Biosciences, Heidelberg, Germany), anti‐CD5 (BioLegend, Koblenz, Germany), anti‐CD20, anti‐CD23, and anti‐CD27 (each BD Biosciences) antibodies. Stained cells were analyzed on a CytoFLEX S flow cytometer (Beckman Coulter, Krefeld, Germany) using CytExpert v2.4 (Beckman Coulter) or FlowJo v10.6.2 (BD Biosciences) software. RNAseq data were retrieved from (Budeus et al, 2021 (link)).
9
Multiparametric Flow Cytometry Analysis
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Cells were harvested and washed with FACS buffer. For surface staining, the 1 × 106 cells in 100-μL single-cell suspension were stained with antibodies for 30 minutes in the dark at 4 °C. The following antibodies were used: live and dead cell stain FVS (BD Biosciences), anti-CD3, anti-CD4, anti-CD8, anti-CD103/ITGAE, anti-CD19, anti-CD27, anti-IgD, anti-CD38, and anti-CD138 (BD Biosciences, San Jose, CA); and anti-CD69, anti-CD18/ITGB2, anti-KLRG1 (BioLegend, San Diego, CA). For intracellular cytokine staining, immune cells were incubated in complete RPIM1640 containing 10% FBS and leukocytes activation cocktail with GolgiPlug (BD Biosciences) at 37 °C for 5 hours, and then surface-stained cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) for 20 minutes at 4 °C. Subsequently, cells were stained with anti-GZMB, anti-GZMK, and anti-IgA (BD Biosciences) for 30 minutes at 4 °C. For staining TFs, cells were fixed and permeabilized with Transcription Factor Buffer Set (BD Biosciences). Briefly, after surface staining, cells were fixed and permeabilized for 50 minutes at 4 °C, then washed and stained with ATF5 (Abcam, Cambridge, UK) for 50 minutes at 4 °C. FCM was carried out using Celesta (BD Biosciences) and analyzed using FlowJo software (version 10.6.2, Tree Star).
10
PBMC Mitochondrial Staining and Flow Cytometry
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Freshly isolated or thawed total PBMCs were pulsed with 20 nM of MitoTracker Green (Invitrogen, Inc.) in pre-warmed complete media (RPMI 1640 supplemented with 10% FBS and 1% L-glutamine) at 37 °C for 30 min. Cells were pelleted at 1,300 RPM for 10 min at RT, resuspended, and chased with 10 ml of pre-warmed complete media for 30 min at 37 °C. Cells were again spun at 1,300 RPM for 10 min, resuspended at 107 cells per 100 μl of PBS containing 0.5% BSA, 5% normal mouse serum, and 5% normal rat serum, and stained for flow cytometry with the following fluorochrome conjugated mouse anti-human monoclonal antibodies: anti-CD3, anti-CD24 (Invitrogen, Inc.), anti-CD19, anti-IgD, anti-CD27, anti-CD38 (BD Biosciences, Inc.). Analysis was performed using a BD LSRII. Sorting was performed on a FACS Aria II. Prior to each sort the FACS AriaII was calibrated with fluorescent beads to achieve a >99% sort purity.
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