For assessment of cell surface phenotypic markers, peripheral blood from the study patients was stained with fluorescein isothiocyanate- (FITC-), phycoerythrin- (PE-), allophycocyanin- (APC-), and peridinin chlorophyll protein- (PerCP-) conjugated murine monoclonal antibodies (mAbs). For dendritic cell detection, Lin cocktail CD3/14/16/19/20/56-FITC, HLA-DR-PerCP, pDC marker CD123-PE, and cDC marker CD11c-APC (BioLegend, USA) were used; for NK, CD3-FITC and CD16+CD56-PE (Simultest™, Becton Dickinson, UK). Isotype controls were obtained from BioLegend. In test tubes containing 50 μl of peripheral blood supplemented with anticoagulant, appropriate amounts of mAbs, according to the manufacturer's recommendations, were added. All samples with labeled antibodies were incubated for 30 min at RT in the dark. After incubation, red blood cells were lysed for 15 min with 160 mM NH4Cl solution at RT in the dark, and then the samples were centrifuged (500×g). Precipitated cells were washed with Cell Wash Buffer (Becton Dickinson, USA) and analyzed by two- or four-color flow cytometry on a FACSCalibur flow cytometer and CELLQuest software after calibration with CaliBRITE beads (BD Biosciences, San Jose, CA, USA). Data for each sample were acquired until 100,000 cells were analyzed.
Hla dr percp
Manufactured by BioLegend
Sourced in United States
HLA-DR-PerCP is a fluorescently labeled antibody that binds to the human leukocyte antigen (HLA) class II molecule, HLA-DR. It is a useful tool for the identification and analysis of HLA-DR-expressing cells, such as antigen-presenting cells, by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti ige, by Merck Group (3 mentions)
Cd63 apc, by BioLegend (3 mentions)
Cd203c pe, by BioLegend (3 mentions)
Cd38 pe, by BioLegend (2 mentions)
Cd123 fitc, by BioLegend (2 mentions)
Facsdiva software, by BD (2 mentions)
Formyl methionyl leucylphenylalanine fmlp, by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Calibrite beads, by BD (1 mentions)
Cell wash buffer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd86 pe, by BD (1 mentions)
Cd19 bv510, by BioLegend (1 mentions)
Cd27 pe cy7, by BioLegend (1 mentions)
Igm fitc, by Thermo Fisher Scientific (1 mentions)
Cd40 pe, by BD (1 mentions)
7 aad staining, by BD (1 mentions)
Cd5 fitc, by Thermo Fisher Scientific (1 mentions)
Cd24 apc, by Thermo Fisher Scientific (1 mentions)
9 protocols using hla dr percp
1
Immunophenotyping of Peripheral Blood Cells
2
Phenotyping of B cell subsets
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For phenotyping of B cells, CD9+ and CD9− B cells, the antibodies used were as follows: CD27-PE-Cy7 (clone 0323, Biolegend), CD38-PE (clone HB7, Biolegend), CD19-BV510 (clone HIB19, Biolegend), CD24-APC (clone SN3 A5-2H1D, eBioscience, San Diego, CA, USA), HLA-DR-PERCP (Clone L243, Biolegend), CD40-PE (Clone 5C3, BD Biosciences), CD86-PE (Clone 2331, BD Biosciences), CD25-PE/Cy5 (Clone M-A251, BD Biosciences), CD69-APC (Clone FN50, Biolegend), IgD-APC-Cy7 (Clone, IA6-2, Biolegend), IgM-FITC (Clone, SA-DA4, eBioscience), CD5-FITC (Clone, L17F12, eBioscience), CD10-APC (Clone, LT10, ImmunoTools, Friesoythe, Germany), CD20-PE-Dy647 (Clone, LT20, ImmunoTools) and CD21-FITC (Clone, LT21, ImmunoTools). Viability was assessed by 7AAD staining (BD Biosciences). All flow cytometric acquisition was performed with FACS Canto II, BD Biosciences. The data analysis was performed using FlowJo V7 (TreeStar Inc., Ashland, OR, USA). The gating strategy for the Bregs and single markers can be found in Supplementary Figure S1 .
3
Basophil Activation Test for Allergy Diagnosis
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The BAT was performed using heparinized venous blood on the same day of blood collection. Basophils in 100 μL of whole blood were stimulated with the same volume of egg extract (ALK-Abello), baked EW (Sigma-Aldrich, Poole, UK), anti-IgE (1 μg/mL, Sigma-Aldrich, Poole, United Kingdom) or N-formyl-methionyl-leucyl-phenylalanine (f-MLP; 1 μM, Sigma-Aldrich) diluted in Roswell Park Memorial Institute medium (GIBCO, Paisley, United Kingdom) with Roswell Park Memorial Institute alone as negative control. Cells were stained with CD123-FITC, CD203c-PE, human leukocyte antigen (HLA)-DR-PerCP, and CD63-APC (Biolegend, San Diego, Calif) and analyzed by flow cytometry using Fortessa with Diva software (BD Biosciences, San Jose, Calif). Data were analyzed with FlowJo software (version 7.6.1; TreeStar, Ashland, Ore) gating basophils as low side scatter/CD203c+/CD123+/HLA-DR-11 (link),17 (link) cells. Basophil activation was expressed as %CD63+ basophils.
4
Basophil Activation Test for Allergy
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Blood was collected in lithium heparin‐containing tubes, and the BAT was performed on the same day of blood collection. Per condition, 100 μL of whole blood was incubated with the same volume of egg extract (ALK‐Abello) or baked EW (Sigma‐Aldrich) diluted in RPMI (GIBCO). Baked EW was prepared as a single batch by heating in a oven at 180°C for 20 min and stored at −80°C thereafter. Anti‐IgE (1 μg/mL; Sigma‐Aldrich) and formyl‐methionyl‐leucylphenylalanine (fMLP, 1 μM; Sigma‐Aldrich) were used as positive controls and RPMI alone as a negative control. Cells were stained with CD123‐FITC, CD203c‐PE, HLA‐DR‐PerCP, and CD63‐APC (all Biolegend) which had been lyophilised in individual tubes as a single batch at the start of the study. Flow cytometry was performed using FACS Fortessa with FACSDiva software (BD Biosciences). The flow cytometry data were analysed using FlowJo software (version 7.6.1; TreeStar). Basophils were gated as SSClow/CD203c+/CD123+/HLA‐DR‐.
11 (link),
17 (link) Basophil activation was expressed as %CD63+ basophils. Individuals with non‐responder basophils were defined as having %CD63+ basophils below 5% to Anti‐IgE and response above 5% to fMLP, and were excluded from the R diagnostic analyses.
11 (link),
17 (link) Basophil activation was expressed as %CD63+ basophils. Individuals with non‐responder basophils were defined as having %CD63+ basophils below 5% to Anti‐IgE and response above 5% to fMLP, and were excluded from the R diagnostic analyses.
5
Multiparametric Flow Cytometry for Cell Surface and Intracellular Markers
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Anti-ICOSL (MIH12), ICOSL-APC, CD16-PE, anti–VCAM-1-PE, anti–E-Selectin-PE, CD27-PE, CD19-APC, CD45-PerCP/Cy5.5, goat anti-rabbit Alexa Fluor 488, goat anti-mouse Alexa Fluor 633, and Alexa Fluor 568 were from Thermo Fisher Scientific. CD19-FITC, IgD-FITC, FoxP3-A647, CD14-FITC, and CD45RA-PE were from BD Biosciences. Antibodies against RCAS1 and β-actin were from Cell Signaling Technology. HLA-DR-PerCP, CD11c-Pacific Blue, CXCR5-BV421, CCR7-APC/Cy7, PD1-PE/Cy7, and ICOS-APC were from BioLegend. BDCA1 (CD1c)-APC, BDCA3 (CD141)-PE, CD4-FITC, and ICAM-1-FITC were from R&D Systems.
6
Phenotypic Characterization of MDSC Subsets
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Extracellular staining with fluorescent antibodies on fresh or frozen PBMC was done as described previously [8 (link)]. At each time point, analyses were performed for gMDSC and mMDSC respectively. The following antibodies were used: gMDSC: CD11b-FITC, CD14-APC, CD15-PerCP, CD66b-PE [14 (link), 17 (link)]; mMDSC: CD11b-FITC, CD14-APC, CD33-PE, HLA-DR-PerCP, PerCP isotype control (all BioLegend, USA). Cells were analyzed on a FACSCalibur (BD, Germany) and analysis of data was done using FlowJo software 7.2.1 (TreeStar, Inc. Ashland, OR). Gating strategies were according to Vollbrecht et al. [8 (link)] and Rieber et al. [14 (link), 17 (link)] for gMDSC. For the gating of mMDSC, we used a PerCP isotype control in order to gate for HLA-DR and our gating strategy was according to Dumitru et al. [2 (link)] (see Additional file 1 : Figure S1). For the statistics, we indicated MDSC as percentage of PBMC in all subgroups because the numbers of monocytes vary substantially in subjects with e.g. chronic viral infections. However, in the dot plots shown in Fig. 2 , frequencies of the described populations are shown in percentages of monocytes.
7
Cell Surface Protein Staining
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The staining for cell surface proteins were performed following standard protocols by incubation of cells with pre-optimized concentrations of antibodies at 4°C for 20 min in FACS buffer (PBS with 2% FCS and 1 mM EDTA). Flow cytometry data were acquired on BD FACS Canto II cytometer and analyzed with FlowJo software version X (Tree Star). Unless otherwise stated all antibodies were supplied by BD Biosciences: CD25 APC (M-A251) or CD25 PECy7 (2A3); CD8 APC (SK1); CD4 V500 (RPA-T4), CD3 PerCPCy5.5 (SK7), CD38 PE (HIT2); CD80 PE (L307.4); CD83 FITC (HB15e); CD86 PECy7 (FUN-1), HLA-DR PerCP (L243); CCR7 PECy7 (G043H7; Biolegends). Appropriate isotypes were used as controls.
8
Multiparametric Flow Cytometry Analysis
9
Basophil Activation Test with Egg Extracts
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The BAT was performed as previously described,
5 (link) using whole blood and 10fold dilutions in Roswell Park Memorial Institute (RPMI) medium from 10,000 to 0.1 ng/mL of egg extract (ALK Abello) or baked egg white (Sigma‐Aldrich) as stimulants. Baked EW was prepared as a single batch by heating in an oven at 180°C for 20 min and stored in small aliquots for single use at −80°C. RPMI medium alone was used as negative control and anti‐IgE (1 μg/mL, Sigma‐Aldrich) and formyl‐methionyl‐leucylphenylalanine (fMLP, 1 μM, Sigma‐Aldrich) were used as positive controls. Basophil activation was measured as the proportion of CD63‐expressing cells or as the stimulation index of mean fluorescence intensity of CD203c following stimulation. CD63‐APC and CD203c‐PE antibodies were used to measure activation and CD123‐FITC, CD203c‐PE, HLA‐DR‐PerCP and CD63‐APC (Biolegend) were used to identify the basophil cell population by flow cytometry using FACS Fortessa with FACSDiva software (BD Biosciences) to acquire and FlowJo software (version 7.6.1; TreeStar) to analyze the data.
5 (link) using whole blood and 10fold dilutions in Roswell Park Memorial Institute (RPMI) medium from 10,000 to 0.1 ng/mL of egg extract (ALK Abello) or baked egg white (Sigma‐Aldrich) as stimulants. Baked EW was prepared as a single batch by heating in an oven at 180°C for 20 min and stored in small aliquots for single use at −80°C. RPMI medium alone was used as negative control and anti‐IgE (1 μg/mL, Sigma‐Aldrich) and formyl‐methionyl‐leucylphenylalanine (fMLP, 1 μM, Sigma‐Aldrich) were used as positive controls. Basophil activation was measured as the proportion of CD63‐expressing cells or as the stimulation index of mean fluorescence intensity of CD203c following stimulation. CD63‐APC and CD203c‐PE antibodies were used to measure activation and CD123‐FITC, CD203c‐PE, HLA‐DR‐PerCP and CD63‐APC (Biolegend) were used to identify the basophil cell population by flow cytometry using FACS Fortessa with FACSDiva software (BD Biosciences) to acquire and FlowJo software (version 7.6.1; TreeStar) to analyze the data.
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