A panel of monoclonal antibodies was developed to enable phenotypic characterization of B and T lymphocyte subpopulations: CD3-FITC, CD4-APC, CD25-BV421, FoxP3-PE, CD19-PeCy7, CD9-BB700 (BD Bioscience, France), CD24-BV510, and CD38-APC-Cy7 (Sony Biotechnology, UK); hematopoietic stem and progenitor cells: CD34-BV421 (Biolegend, France), cKit-APC-H7, CD135-PE, lineage-FITC, Sca-1-BV510, CD16/32-PerCPCy5.5, CD127-APC (BD Bioscience), and TLR-4-PeCy7 (Biolegend); and dendritic cells: CD11c-PeCy7 (eBioscience, France), CMH-II-FITC, Siglec-H-BV510, CD11b-APC-H7, and CD103-PerCP5. For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences). For in vitro human B cell differentiation experiments, cells were stained with Fixable Viability Dye eFluor 450 to identify dead cells, followed by staining with CD25-BV605 (Biolegend), CD19-BUV395, CD9-FITC, CD27-BUV737, CD38-BV711 and intracellular IL-10-PE (BD Bioscience). Cells were analyzed on a Canto II flow cytometer (BD Biosciences). Data were acquired using Diva 8.0 software and analyzed with FlowJo X (TreeStar, Williamson Way, Ashland, USA). Fluorescence minus one staining controls were used for all panels, and dead cells were removed using viability staining.
Cd16 32 percpcy5
Manufactured by BD
Sourced in United States
The CD16/32-PerCPCy5.5 is a fluorochrome-conjugated antibody used for the detection and analysis of CD16 and CD32 expression on cells. It is designed for flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd11c pe cy7, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 450, by BioLegend (1 mentions)
Cd135 pe, by BD (1 mentions)
Cd127 apc, by BD (1 mentions)
Fitc cd9, by BD (1 mentions)
Cd25 bv605, by BioLegend (1 mentions)
Lineage fitc, by BD (1 mentions)
Cd38 bv711, by BD (1 mentions)
Cd34 bv421, by BioLegend (1 mentions)
Cd19 buv395, by BD (1 mentions)
Cd27 buv737, by BD (1 mentions)
Foxp3 pe, by BD (1 mentions)
Cd25 bv421, by BD (1 mentions)
Cd19 pe cy7, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Cd3 fitc, by BD (1 mentions)
Cd4 apc, by BD (1 mentions)
Cd64 apc, by BD (1 mentions)
Mhcii fitc, by BD (1 mentions)
3 protocols using cd16 32 percpcy5
1
Comprehensive Immune Cell Profiling
2
Multiparametric Flow Cytometry Analysis
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Peripheral blood, spleens, and mesenteric lymph nodes (mLN) were collected 24 h after CLP surgery or sham surgery to obtain single cells for flow cytometry. Cells were stained for 30 min at 4°C with the following antibodies: CD45-Percp, CD3-FITC, CD4-Percp, CD8-APC, B220-PE, CD11c-APC, CD11b-FITC, F4/80-PE, and Ly6G-PE. Following incubation, red blood cells were lysed and washed 3 times with a FACS buffer before collecting data. Immunophenotyping analysis of the BMDMs was conducted using a flow cytometry technique. Briefly, 100 μl of cell suspension was incubated for 5 min with 5 μl of Fc blocker (Innovex, USA). Then, 1 μl of monoclonal antibodies (CD11b-FITC, F4/80-PE, F4/80-FITC, MHCII-FITC, CD80-PE, CD80-APC, CD16/32-Percp-cy5.5, and CD64-APC, BD, USA) were added and incubated at 4°C for 30 min and washed 3 times with the FACS buffer. All samples were analyzed using a FACSCalibur flow cytometer (BD, USA).
3
Bone Marrow Cell Isolation and Phenotyping
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Thawed bone marrow was resuspended in 1mL RBC lysis buffer and incubated for 1 minute at 25°C. PBS (25mL, Gibco) was added to suspension and centrifuged prior to resuspension in thawing medium.
Five million cells were plated on a round-bottom 96-well plate. Samples were centrifuged at 400 rcf for 5 minutes and incubated for 20 minutes in 30uL 0.2μg/uL (6μg) of CD16/32 PerCP-Cy5.5 (BD Biosciences, USA). Samples were washed and then incubated in 30μL of a 6-colour panel and Ms CD16/32 FACS Block (see Table S1). The gating strategy is shown in Figure 5A. Single-colour compensation controls were either prepared using remaining bone marrow (500,000 cells/well) or using rat/hamster compensation beads (BD anti-rat, anti-hamster Ig κ/negative control set, BD Biosciences).
Both compensation controls and samples were washed twice with 200μL FACS buffer (2% FCS (Gibco) in PBS (Gibco)), then resuspended in 200μL FACS buffer and transferred to 5mL FACS tubes for analysis on a BD FACSAria Fusion (SORP) at the Translational Research Institute (Brisbane, Australia). Percentages of hematopoietic stem cells (HSPCs), common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) were quantified (Figure 5A).
Five million cells were plated on a round-bottom 96-well plate. Samples were centrifuged at 400 rcf for 5 minutes and incubated for 20 minutes in 30uL 0.2μg/uL (6μg) of CD16/32 PerCP-Cy5.5 (BD Biosciences, USA). Samples were washed and then incubated in 30μL of a 6-colour panel and Ms CD16/32 FACS Block (see Table S1). The gating strategy is shown in Figure 5A. Single-colour compensation controls were either prepared using remaining bone marrow (500,000 cells/well) or using rat/hamster compensation beads (BD anti-rat, anti-hamster Ig κ/negative control set, BD Biosciences).
Both compensation controls and samples were washed twice with 200μL FACS buffer (2% FCS (Gibco) in PBS (Gibco)), then resuspended in 200μL FACS buffer and transferred to 5mL FACS tubes for analysis on a BD FACSAria Fusion (SORP) at the Translational Research Institute (Brisbane, Australia). Percentages of hematopoietic stem cells (HSPCs), common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) were quantified (Figure 5A).
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