Macrophages derived from the bone marrow and lung homogenates of BALB/c mice were evaluated using flow cytometry. The lung cells and BMMs were treated with 10% mouse serum for 30 min. After treatment, the cells were washed with 200 μL PBS/azide. After centrifugation, the macrophages were incubated for 30 min with FITC anti-CD206 (Clone MR5D3 – Santa Cruz Biotechnology), anti-CD86 PE (Clone GL1-eBioscience Inc., San Diego, CA, USA), anti-CD11b PerCP (Clone M1/70 – BD Biosciences Pharmingen, San Jose, CA, USA), and anti-F4/80 APC (Clone BM8 - eBioscience, Inc., San Diego, CA, USA) antibodies. Meanwhile, the BMMs were labeled with anti-CD206 FITC (MR5D3 – Santa Cruz Biotechnology), anti-CD86 PE (Clone GL1 – eBioscience Inc., San Diego, CA, USA), anti-MHCII PerCP (Clone M5/114.15.2 – BioLegend), and anti-F4/80 APC (Clone BM8 – eBioscience, Inc. San Diego, CA, USA) antibodies. For intracellular staining, cells were first treated with PermWash (BD PermWashTM) and then incubated with anti-TNF-α FITC (Clone MP6-XT22). After the addition of 200 μL PBS/azide and further centrifugation, the cells were treated with PERM FIX (BD Cytofix/CytopermTM) for 20 min at 2–8°C. They were then washed and resuspended in 200 μL PBS/azide. A total of 50,000 events were acquired using the BD FACS Verse flow cytometer (UFG). Data were analyzed using FlowJo software, version 8.7.
Anti cd86 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Anti-CD86-PE is a fluorescently labeled antibody that specifically binds to the CD86 cell surface marker. It is commonly used in flow cytometry applications to identify and analyze cells expressing the CD86 antigen.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (7 mentions)
Anti cd80 pe, by Thermo Fisher Scientific (6 mentions)
Anti cd11c fitc, by Thermo Fisher Scientific (5 mentions)
Anti cd80 fitc, by Thermo Fisher Scientific (5 mentions)
Anti cd40 pe, by Thermo Fisher Scientific (4 mentions)
Anti mhcii fitc, by Thermo Fisher Scientific (4 mentions)
Anti cd25 pe, by Thermo Fisher Scientific (4 mentions)
Anti cd11c apc, by Thermo Fisher Scientific (3 mentions)
Anti mhc 2 pe, by Thermo Fisher Scientific (3 mentions)
Anti cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Anti cd40 fitc, by BD (2 mentions)
Anti hla dr pe, by Thermo Fisher Scientific (2 mentions)
Pecy7 anti cd45, by BD (2 mentions)
Anti cd11c alexa700, by Thermo Fisher Scientific (2 mentions)
Lps free fbs, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Anti hla dr fitc, by Thermo Fisher Scientific (2 mentions)
39 protocols using anti cd86 pe
1
Comparative Analysis of Murine Macrophage Phenotypes
2
Flow Cytometric Analysis of Immune Cells
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The population of each phenotype of immune cells in different groups was evaluated by using flow cytometric analysis as described previously [30 (link)]. In brief, splenocytes were stained with fluorescent antibodies, including anti-CD4-FITC, anti-CD25-PE, anti-Foxp3-PerCP, anti-CD11c-APC, anti-MHCII-FITC, anti-CD86-PE, anti-CD40-PE, anti-CD4-PE (eBiosciences, San Diego, USA), anti-IL-4-APC, anti-CD68-FITC, and anti-CD206-PE (BioLegend, San Diego, USA), according to the manufacturer’s instruction. The FlowJo software was used to analyze the percentages of various phenotypes of immune cells.
3
Visualizing Skin Dendritic Cells
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GFP-MHC-II mice received 10 μg of CTB or PBS i.d. in the ear. After 12, 24 or 72 h, epidermal sheets were obtained, stained with anti-CD86-PE (eBioscience), mounted with VectaShield (Vector Laboratories) and sealed. The images were obtained with a Leica TCS SP8x Confocal Microscope (Wetzlar, Germany) and analyzed with Leica Application Suite Advanced Fluorescent Lite software (Leica Microsystems, Mannheim, Germany). Alternatively, C57BL/6 mice received 10 μg of CTB or PBS i.d. in the ear. After 24, 72 h, or 7 days, mice were sacrificed to collect SDLN and skin. Tissues were processed and stained to be analyzed by flow cytometry.
4
Multiparametric flow cytometry analysis
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Cell surface molecules were analysed by a FACSCalibur® flow cytometer (Becton Dickinson). Cells were incubated for 10 min at 4°C with relevant or control antibodies in 100 µl total staining volume in PBS-BSA. After staining, the cells were washed and resuspended in 300 µl of PBS-BSA. The following antibodies were used for the staining of human monocytes, T cells and dendritic cells:, anti-CD1c-FITC [anti-BDCA-1] (Miltenyi), anti-CD11b-PE [Mac-1] (BD Pharmingen), anti-CD14-APC (BD Pharmingen), anti-CD40-FITC (BD Pharmingen), anti-CD45RA-APC (BD Pharmingen), anti-CD86-PE (eBioscience), anti-HLA-DR-CyChrome (BD Pharmingen). The following antibodies were used for the staining of murine dendritic cells and T cells: CD4-AF647, vα2-Bio, SA-PacificBlue, CD11c-FITC, CD86-PE, MHC II was measured by a combination of biotin-labeled I-Ab/SA-APC (all BD Pharmingen). To measure proliferation, T cells were labeled with the fluorescent dye CFSE (Molecular Probes).
5
Monocyte Subsets and Co-Stimulatory Molecules Analysis
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The ex vivo frequency of monocyte subsets and expression of HLA-DR, CD80 and CD86 was determined using PBMCs from HC, HAM/TSP patients and HS. Cells were stained with monoclonal antibodies (anti-CD14-FITC, anti-CD16-PE-Cy5, anti-HLA-DR-PE, anti-CD80-PE e anti-CD86-PE, from eBioscience, San Diego, CA or R&D Systems, Minneapolis, MN) for 20 minutes at 4°C. PBMCs were washed with PBS and then fixed with 2% paraformaldehyde. Cells were then analyzed on the flow cytometer (II FacsCanto, BD Biosciences, San Jose, CA). Analysis was performed using FlowJo software version 7.6 (TreeStar, Ashland, OR). The monocyte population was selected based on size and cell granularity and then subdivided into classical (CD14++CD16-), intermediate (CD14+CD16+) and non-classical monocytes (CD14+CD16++).
6
Immunophenotyping of Macrophage Polarization
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Antibodies such as anti-iNOS-Alexa Fluor 488 (Cat. number 53-5920), anti-CD86-PE (Cat. number 12-0862), and anti-CD11c-PE (Cat. number 12-0114) were purchased from eBioscience (California, USA). Anti-CD206-FITC antibody was purchased from Biolegend (California, USA, Cat. number 141703). Anti-Arg1-PE antibody was purchased from R&D systems (Minnesota, USA, Cat. number IC5868P). For pathway research, FCM was used as previously described [15 , 16 (link)]. Anti-phospho-P65 antibody (Cat. number 3033) and anti-P65 antibody (Cat. number 8242) were purchased from Cell Signaling Technology (Massachusetts, USA). The primary antibody against RAGE for FCM was purchased from Abcam (Cambridge, UK, Cat. number ab3611). The secondary antibody conjugated with Alexa Fluor 488 was purchased from Beyotime (Jiangsu, China, Cat. number A0423). The antibody for blocking and neutralizing RAGE was purchased from R&D systems (Minnesota, USA, Cat. number AF1179). The NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) was purchased from Abcam (Cambridge, UK, Cat. number ab141406). Results were acquired by a BD Canto II flow cytometer (BD Biosciences, USA) and analyzed by the FlowJo software (Tree Star, USA).
7
Dendritic Cell Isolation and Stimulation
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Bone marrow was harvested and cultured as previously described[23 (link)]. On day six of the culture the cells were harvested from the plates and semi-adherent cells removed. The cells were spun at 400g and re-suspended at a concentration of 1×106 cells/ml in DC medium (RPMI 1640 supplemented with 10% LPS-free FBS (Gibco, Paisley, UK) 1% Penicillin/streptomycin and 50mM Beta-mercaptoethanol (Sigma Aldrich, Dorset UK)). The cells were stimulated overnight with LPS (100ng/ml) or T. muris antigen (5μg/ml). After 24 hours the cells were harvested and prepared for flow cytometry. FcR were blocked by incubating cells in anti-CD16/32 (2μg/ml) for 15 minutes. Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson), anti-CD11c Alexa700 (1μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-CD86 PE (1 μg/ml) (all E Bioscience) and acquired using a LSRII flow cytometer (Becton Dickinson Biosciences, Oxfordshire). Data was analysed using Flow Jo flow cytometry analysis software (Tree Star, Inc, Oregon, US).
8
Lung DC Phenotyping in Treated Mice
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The lungs were isolated from treated mice and digested as previously described (16 (link)). Tissues were dissociated and single cell suspensions were obtained. The following antibodies were purchased from eBioscience (San Diego, CA, USA): 1:800 anti-CD80 Pe, 1:600 anti-CD86 PE, and isotype control antibodies. Lung DCs were labeled with antibodies. After 30 min at 4°C, cells were washed twice and fixed in 2% paraformaldehyde (PFA; Alfa Aesar, Haverhill, MA, USA) diluted in PBS-BSA 0.2% for 15 min at 4°C. Cells were washed, resuspended in PBS-BSA 0.2%, and analyzed by flow cytometry. Finally, samples were collected and analyzed on a FACSCalibur flow cytometer (BD Immunocytometry Systems, Franklin Lakes, NJ, USA).
9
BAL Immune Cell Phenotyping and Signaling
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Bronchoalveolar lavage (BAL) samples were obtained by using a standardized protocol of fiberoptic bronchoscopy and filtered through nylon gauze (24 (link)). After centrifuged for 10 min at 400 g at 4°C, cells were resuspended in fluorescence-activated cell sorter (FACS) buffer (PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA). Cells were stained with conjugated monoclonal antibodies to detect membrane markers. To further examine intracellular markers, cells were subsequently fixed with 4% paraformaldehyde (PFA) fixation for 40 min and permeabilized with Fixation and Permeabilization Solution (BD) for 20 min, and stained with conjugated antibodies to detect intracellular markers. The following human antibodies were applied for BAL samples: anti-CD68 FITC (eBiosciences), anti-CD86 PE (eBiosciences), anti-CD163 APC (eBiosciences). The cells from BAL were not stained with anti-CD16/CD32 antibody for blocking Fc receptors in this analysis. Besides, MH-S cells were treated with stimulus, and were staining the following mouse antibodies: anti-p-Stat5 FITC (eBiosciences) and anti-p-Stat3 PE (eBiosciences). The staining cells were measured through Canto Flowcytometer (BD Biosciences). Data were analyzed using FlowJo 7.6 (BD) software. Isotype controls and fluorescence minus one control were used to set gates.
10
Multiparameter Flow Cytometry Analysis
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The following antibodies were used for cell surface marker evaluation: anti-CD4-PE-Cy5, anti-CD69-PE, anti-CD127-PE-Cy7, anti-CD20-PE-Cy7, anti-CD86-PE, anti-CD80-FITC (eBioscience, San Diego, CA), anti-CD25-ECD, anti-HLA-DR-ECD, and anti-CD11c-PC5 (Beckman Coulter, Brea, CA). Primary human cells or Ramos cells were suspended in FACS buffer (1× PBS, 1% BSA, 0.1% NaN3) and incubated for 20 minutes with panels of the indicated antibodies at 4°C. After washing, cells were analyzed in a Navios flow cytometer (Beckman Coulter, Brea, CA) and data was analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR). Gates used for analysis were set using appropriate isotype controls.
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