Multi-color flow cytometry analysis was performed on PBMCs from all time points by staining for 30 minutes at 4°C with CD3-V450, CD8-FITC or APC, ICOS-PE, HLA-DR-PerCP-Cy5.5, CD25-PE-Cy7, CD45RA-PerCP-Cy5.5, CD62L-FITC, CD127-V450, PD-1-PE, Tim-3-AF700, CD4-APC-Cy7 (BD Biosciences, San Jose, CA), CCR7-PE-Cy7 (R&D Systems, Minneapolis, MN), CTLA-4-FITC (LSBio, Seattle, WA) and FoxP3-APC (eBioscience, San Diego, CA) for T cells. For natural killer (NK) cells, CD3-V450, CD16-APC-Cy7, CD56-PE-Cy7 and Tim-3-AF700 (BD) were used. For myeloid-derived suppressor cells (MDSCs), CD33-PE, CD11b-APC-Cy7, HLA-DR-PerCP-Cy5.5, CD14-V450 and CD15-APC (BD) were used. 1×105 cells were acquired on an LSRII (BD), and data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR). The appropriate isotype controls were used, and dead cells were excluded from the analysis.
Hla dr percp cy5
Manufactured by BD
Sourced in United States, United Kingdom
HLA-DR-PerCP-Cy5.5 is a fluorescently-labeled antibody that binds to the HLA-DR antigen expressed on the surface of certain cells. It is designed for use in flow cytometry applications to identify and quantify HLA-DR-positive cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pd 1 pe, by BD (6 mentions)
Facscanto 2, by BD (6 mentions)
Cd11b apc cy7, by BD (5 mentions)
Cd14 pe cy7, by BD (5 mentions)
Cd45 fitc, by BD (5 mentions)
Cd68 fitc, by Thermo Fisher Scientific (4 mentions)
Cd206 pe, by BD (4 mentions)
Cd14 v450, by BD (3 mentions)
Cd40 pe, by Bio-Rad (3 mentions)
Cd11c pe cy7, by BD (3 mentions)
Cd4 pacific blue, by BioLegend (3 mentions)
Cd11b pe, by Thermo Fisher Scientific (3 mentions)
Cd86 pe cy7, by BD (2 mentions)
Cd69 pe, by BD (2 mentions)
Cd8 fitc, by BD (2 mentions)
Cd25 pe cy7, by BD (2 mentions)
Cd62l fitc, by BD (2 mentions)
Cd4 apc cy7, by BD (2 mentions)
Cd45ra percp cy5, by BD (2 mentions)
Cd127 v450, by BD (2 mentions)
29 protocols using hla dr percp cy5
1
Multiparametric Flow Cytometry Analysis
2
Multi-Dimensional Immune Profiling
3
Phenotypic and Functional Analysis of DCs
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The phenotype of DCs was assessed by flow cytometry in a BD FACS Aria (Becton Dickinson, USA) using corresponding monoclonal antibodies labeled with fluorochromes (CD3-Pacific Blue, CD14-FITC, HLA-DR-PerCP-Cy5.5, CD11c-PE, CD86-PE-Cy7, and CD83-APC; Becton Dickinson, USA) according to the manufacturer’s recommendations. Examples of the used gates are shown in additional files (see Additional file 2 ).
The criterion for functional activity of DCs was their susceptibility to receptor-mediated endocytosis by FITC-dextran capture (Sigma, USA). Briefly, the cells were incubated with FITC-dextran (1 μg/ml) in the complete medium at 4 and 37 °C. Dextran became bound to the surface receptors at 4 °C, and the bound dextran penetrated into the cells at 37 °C (endocytosis).
The criterion for functional activity of DCs was their susceptibility to receptor-mediated endocytosis by FITC-dextran capture (Sigma, USA). Briefly, the cells were incubated with FITC-dextran (1 μg/ml) in the complete medium at 4 and 37 °C. Dextran became bound to the surface receptors at 4 °C, and the bound dextran penetrated into the cells at 37 °C (endocytosis).
4
NK Cell Phenotyping Post-Transplant
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Frozen PBMC and BAL samples were thawed rapidly and the viability determined by trypan blue exclusion. Antibodies used in this study included the following: CD56 Brilliant Violet (BV) 421, CD3 PerCP, KIR3DL1-APC, CD57-FITC (all from Becton Dickinson Biosciences, NJ, USA), NKG2A-PE, NKG2C-AlexaFluor 700 (both from R&D systems), KIR2DL1/2DS1 PECy7, KIR2DL2/2DL3/2DS2 PECy5.5 (all from Beckman Coulter), and KIR3DL2-biotin (courtesy of K.J. Malmberg, Karolinska University Hospital, Stockholm, Sweden) followed by Streptavidin BV 605. There were additional PBMC and BAL samples available for one high-risk recipient at 9 months posttransplant, and further phenotyping was performed by antibodies to CD2-FITC, CD69-PE, HLA-DR PerCP Cy5.5, Ki67 PECy7, CD16 BV 605, and CD103 BV711 (all from Becton Dickinson Biosciences, NJ, USA), ILT2 APC (Beckman Coulter), as well as antibodies to identify NKG2C + NK cells as above (CD3 PerCP, CD56 BV 421, and NKG2C-AlexaFluor 700). All samples also included a viability dye (Live/Dead Fixable Near-IR, Thermofisher). Flow cytometry analysis was performed using a Becton Dickinson LSRFortessa (NJ, USA). Samples were gated on single, live lymphocytes, followed by gating on CD3 -CD56 + and analyzed using FlowJo software (Treestar, San Carlos, USA).
5
Comprehensive Immune Cell Profiling Protocol
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PBMCs were thawed, washed and incubated with Gamunex and EMA, then stained with a cocktail of lineage (Lin) markers (CD3, CD19, CD56)-Brilliant Violet 605 (BioLegend, BD-Biosciences), CD14-Brilliant Violet711, CD11c-PE-Cy7 (BioLegend), CD45-V500, CD123-BV421, HLA-DR-PerCP-Cy5.5, CD15-FITC, CD11b-APC-Cy7, CD33-Alexa Fluor 700 (BD-Biosciences), and CD124-APC (R&D Systems) using EMA to identify dead cells.
To characterize Tregs, we used the same panel as before [23 (link)], staining PBMCs forCD3 (OKT3 supernatant) with Pacific Orange-conjugated secondary antibody (Invitrogen) followed by staining for CD4-Pacific Blue, CD45RA-Alexa Fluor-700, CD8-Peridinin-chlorophyll protein (PerCP), CD279-PerCP-Cy5.5, CD127-Alexa Fluor-647 (Bio legend), CD25-APC-Cy7 (BD Biosciences) and intracellular staining for FoxP3-PE (Bio legend). All samples were measured using a BD LSRII (BD Biosciences) immediately after staining.
To characterize Tregs, we used the same panel as before [23 (link)], staining PBMCs forCD3 (OKT3 supernatant) with Pacific Orange-conjugated secondary antibody (Invitrogen) followed by staining for CD4-Pacific Blue, CD45RA-Alexa Fluor-700, CD8-Peridinin-chlorophyll protein (PerCP), CD279-PerCP-Cy5.5, CD127-Alexa Fluor-647 (Bio legend), CD25-APC-Cy7 (BD Biosciences) and intracellular staining for FoxP3-PE (Bio legend). All samples were measured using a BD LSRII (BD Biosciences) immediately after staining.
6
Multiparameter Flow Cytometry Analysis
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Cells were stained according to the manufacturer’s recommendations with the following antibodies alone or in varying combinations, depending on the experiment: CD14-FITC, CD4-APC and CD45RA-PE (all Immunotools, Friesoythe, Germany), CD40-PE (AbD Serotec, Kidlington, UK), CD86-V450, HLA-DR-PerCPCy5.5, CD1a-PE and CD3-APC/Cy7 (all BD). For surface marker staining, PBS containing 1% FCS and 5mM EDTA (Sigma Aldrich) was used as staining buffer. For intracellular cytokine staining with IFNγ-PerCPCy5.5 (eBioscience), IL17A-PE and IL10-Brilliant Violet 421 (both BioLegend), monensin solution (BioLegend) was added to cell cultures for 6 hours before harvesting. Once harvested, cells were fixed and permeabilized using the fixation and permeabilization buffer set from BioLegend. Staining with FoxP3-Alexa Fluor 647 (BD) was performed with FoxP3 staining buffer set (eBioscience). The samples were analyzed by flow cytometry on a BD FACS Canto II.
7
Comprehensive Immunophenotyping of T Cell Subsets
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Blood was collected and processed within 4 hours maximum. An ammonium chloride-based lysing reagent (BD Pharm Lyse, BD Biosciences) was used for erythrocyte deletion of 1ml of blood. After washing, cells were suspended in PBS and stained with Aqua Dye (Invitrogen) for cell viability. Cells were washed again, suspended in staining buffer and divided in four tubes. The four different panels assessed contained some common and some specific antibodies. Common antibodies were: CD3-eFluor 605, CD4-Alexa700 (eBioscience, San Diego, CA), CCR7-Horizon PE-CF594, CD38-Brillant Violet 421, HLA-DR-PerCP-Cy5.5 and CD11c-PE-Cy7 (BD Biosciences). Specific for each panel were: 1) CCR2-PE, CCR5-APC-Cy7, CXCR6-APC (R&D Systems Inc.) and CXCR3-FITC (BioLegend); 2) CD49d (α4)-FITC, β7-APC, CCR9-PE (BD Biosciences) and CD29 (β1)–APC-Cy7 (BioLegend); 3) CD103-FITC, CD54-APC, CD49a (α1)-PE and CD29–APC-Cy7 (BioLegend); 4) CD18-APC, CLA-FITC (BD Biosciences) and CCR10-PE (BioLegend). Cells were acquired using a BD LSRFortessa SORP flow cytometer (Flow Cytometry Platform, IGTP) and analyzed with FlowJo 9.3.2 software (TreeStar). Gates were drawn based on fluorescence minus one-controls and isotypes, and CD3+ CD4- phenotype was considered CD8+ T cells.
8
Comprehensive Immune Cell Phenotyping
9
Monocyte-to-Dendritic Cell Differentiation Assay
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PBMCs were collected using a ficoll gradient (Ficoll-Paque Plus, GE Healthcare) and monocytes were isolated by a CD14 positive selection (Miltenyi, Bergisch Gladbach, Germany). Monocytes were split into five experimental groups: (1) Negative Control (no cytokines), (2) GM–CSF (Sanofi) + IL-4 (Cell Genix) at 1000 U/ml, 3) Recombinant IL32α (R&D Systems) at 100 ng/ml, 4) Recombinant IL32β (R&D Systems) at 100 ng/ml, 5) Recombinant IL32γ (R&D Systems) at 100 ng/ml, and cultured using Cell Genix Media to yield immature DCs at day 5. Immature DC were harvested and surface stained for flow cytometry analysis. Cell surface markers were observed on the double positive, HLA-DR and CD86 population of cells. Antibodies used included CD80 FITC (BD, Clone L307.4), Mouse IgG1 FITC (Beckman Coulter PN IM0639U), CD86 Pe-Cy7 (BD, Clone FUN-1), HLA-DR PerCpCy5.5 (BD, Clone G46-4), CD1B APC (BioLegend, Clone SN13), CD14 APC-Cy 7 (BD, Clone MφP9), CD68 BV 711 (BD, Clone Y1/82A), Mouse IgG2B BV 711 (BD, Clone 27-35), and Zombie Aqua Viability Dye BV 510 (BioLegend).
10
Flow Cytometric Analysis of HMDM Phenotypes
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Flow cytometric analysis was performed as described previously [10 (link),32 (link)]. Briefly, after 48 h of treatments, HMDMs were washed, detached in 2.5 mM EDTA solution on ice and blocked with BD Fc Block™Pure (564220, BD Biosciences, San Jose, CA, USA) for 10 min at room temperature. HLA-DRPerCP-Cy5.5 (552764; Clone: G46-6, BD Biosciences), CD80 BB51 (565008; Clone: L207.4, BD Biosciences), CD163PE-CF594 (562670; Clone: GHI/61, BD Biosciences) or CD14 APC (555399; Clone: M5E2, BD Biosciences) antibodies were added in each sample and incubated on ice for 30 min according to manufacturers’ suggestions. HMDMs were washed and fixed with ice-cold 1% PFA solution, stored on ice, and analysed with a BD LSRFortessa™ Cell Analyzer and the BD FACSDiva v8.0.1 software (BD Biosciences). To quantify the surface marker expression, median fluorescence intensities of singlet cells were used.
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