Markers used to determine monocyte and/or macrophage subsets comprised: FITC-F4/80 (Serotec) and PE-CD16/32, PE-CD11c, PE-CD40, PE-CD284, PE-CD206, PE-CD23(eBioscience). The cells were stained with surface markers and recorded using BD Accuri flow cytometry (BD Biosciences) and analyzed as previously described17 (link),30 (link),58 (link),59 (link).
Cd40 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD40 PE is a fluorochrome-conjugated antibody that binds to the CD40 receptor, which is expressed on the surface of various immune cells, including B cells, dendritic cells, and macrophages. This product can be used in flow cytometry and other immunoassays to identify and analyze CD40-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Cd86 pe, by Thermo Fisher Scientific (3 mentions)
Cd80 fitc, by Thermo Fisher Scientific (3 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (3 mentions)
Cd11c pe, by Thermo Fisher Scientific (2 mentions)
Cd206 pe, by Thermo Fisher Scientific (2 mentions)
Cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Cd25 pe, by Thermo Fisher Scientific (2 mentions)
Cd16 32 pe, by Thermo Fisher Scientific (1 mentions)
F4 80 fitc, by Bio-Rad (1 mentions)
Accuri, by BD (1 mentions)
Ifn γ pe, by Thermo Fisher Scientific (1 mentions)
Il 10 pe, by Thermo Fisher Scientific (1 mentions)
Il 17 pe, by Thermo Fisher Scientific (1 mentions)
Cd8a pe, by Thermo Fisher Scientific (1 mentions)
Anti cd11b alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Cd11c fitc, by Thermo Fisher Scientific (1 mentions)
Mhc 1, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd16 cd32, by Thermo Fisher Scientific (1 mentions)
11 protocols using cd40 pe
1
Monocyte and Macrophage Subset Analysis
2
Multiparameter Flow Cytometry Analysis
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On day 28 p.i., mice were sacrificed and spleens were removed under aseptic conditions. MNCs were prepared as above described stained for 20 minutes at RT in 1% BSA‐PBS buffer with the following panel of antibodies: FITC‐CD4 (eBioscience, San Diego, CA, USA) and PE‐CD25 (eBioscience), Alexa Fluor 488‐anti‐CD11b (eBioscience) and PE‐CD16/32, PE‐CD206, PE‐CD11c, PE‐CD40, PE‐CD8a, PE‐CD14, PE‐CD200 (eBioscience). For intracellular staining, MNCs were stained for 20 minutes at RT in 0.3% saponin/1% BSA‐PBS buffer with the following panel of antibodies: FITC‐CD4 and PE‐IL‐10, PE‐transforming growth factor‐β (TGF‐β), PE‐IFN‐γ, PE‐IL‐17 (eBioscience), Alexa Fluor 488‐anti‐CD11b and PE‐IL‐12, PE‐chemokine receptor7 (CCR7) (eBioscience). At least 10 000 events were collected using flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and data were analysed using CellQuest software (BD Biosciences).
3
Isolation and Phenotyping of Adipose Tissue Lymphocytes
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The C57BL/6 mice at the age of 18–22 weeks were killed and removed the epididymal adipose tissues and minced it into small pieces (∼2 mm). Then the pieces of adipose tissues were collected and incubated them at 37°C for about 2 hours in collagenase II with gentle stirring. The digested tissues were centrifuged at 500 g for 5 min. The resultant pellet containing the total lymphocytes were re-suspended in PBS and filtered it through a 100 M mesh, and we finally re-suspended them in 100 μl PBS supplemented with 5% FBS. All these cells were incubated with FITC-CD11c, PE-CD40, CD80, CD86, MHCI, MHCII (eBioscience, San Diego, CA) conjugated antibodies or respective isotype controls for 30 min at 4°C. All the staining was performed according to manufacturers' protocol. Flow cytometry was performed using FACS Calibur instrument (Becton Dickinson) and WinMDI 2.8 software was used to analyze the data.
4
Isolation and Characterization of Splenic Immune Cells
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Cells were isolated from spleens using mechanical disruption followed by red blood cell (RBC) lysis. Fc receptors of cells were blocked with anti-mouse CD16/CD32 (eBioscience, San Diego, CA) for 15 minutes at 4°C prior to antibody staining. Then, cells were incubated for 30 min at 4°C with the following antibodies: CD19-FITC (eBioscience), CXCR5-APC (BD Biosciences, San Jose, CA), and CD40-PE (eBioscience). After washing twice with PBS containing 1% FBS, cells were characterized using a FACSVerse cytometer (BD Biosciences). Data were analysed with FlowJo (Tree Star, version 10.0.7).
5
Regulatory T cell and Dendritic Cell Analysis
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For Treg analysis, the isolated cells or cultured cells were hybridized with anti‐CD4‐FITC and anti‐CD25‐APC (both from eBioscience, San Diego, CA) for about 30 minutes at 4°C and then stained with anti‐Foxp3‐PE (eBioscience) after fixation and permeabilization following the instructions. For analysis of the characterization of cultured DCs, the cells were stained with CD11c‐APC (BD Biosciences), MHC‐II‐FITC (BD Biosciences), CD40‐PE (eBioscience) or CD86‐PE (eBioscience) for 30 minutes. FACSCalibur (BD Immunocytometry Systems) and Flowjo software (TreestarInc) were used for cell detection and data analysis.
6
Generating Bone Marrow-Derived Dendritic Cells
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BMDC were generated from bone marrow progenitor cells as described previously43 (link). Briefly, progenitor cells were obtained by flushing tibia and femur from BALB/c mice and cultured at 37 °C under 5% CO2 in Iscove’s modified Dulbecco’s media supplemented by FCS (10%), gentamycin (50 µg/ml), glutamine (2 mM), β-mercaptoethanol (50 µM) and 10% of supernatant from a granulocyte–macrophage colony-stimulating factor (GM-CSF)-expressing J588 myeloma cell line for 10 days. On day 10, cells were stimulated by bacteria (ratio bacteria/cell: 10:1). After 24 h stimulation, BMDC were harvested, washed and stained using mAbs anti-CD11c PE-Cy7, CD40 PE, CD80 FITC, CD86 PE-Cy5, MHCII APC (eBioscience, San Diego, CA, USA) and acquired on BD FACS Canto II (Becton Dickinson).
7
Multiparametric Flow Cytometry Analysis
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Cells of MLN (5 × 105) were washed in PBS + 1% BSA and stained with anti-mouse CD3-PE, CD4 FITC, CD8 PE, F4/80 FITC, CD40 PE, CD206 PE, CD25 PE, CD11c PE-Cy5, NK FITC, B220 FITC antibodies (eBioscience) in FlowCytometry Staining Buffer (eBioscience) for 1 h at 4 °C in the dark. After repeated washing in PBS + 1%BSA, cells were resuspended in PBS and immediately detected by CyFlow Space (Partec, Görlitz, Germany) and analysed by FlowMax software (Partec). Prior to intracellular cytokine staining, cells (1 × 106) were stimulated with phorbol myristate acetate (PMA, 100 ng/ml) and ionomycin (400 ng/ml) (both from Sigma-Aldrich) in the presence of Brefeldin A (eBioscience) (5 µM) for 4 h, stained with anti-mouse CD5 FITC and anti-mouse CD19 PE-Cy5 (eBioscience) antibody, fixed in 2% paraformaldehyde, permeabilized with Permeabilization buffer (eBioscience) and then stained for the intracellular cytokines with the anti-mouse IL-10 PE (eBioscience).
8
Multiparameter Flow Cytometry Analysis
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Antibodies used for analysis were anti-human CD11c APC, CD80 FITC, CD83 FITC, HLA-DR FITC, CD40 PE, CD86 PE, TLR-2 PE, CXCR4 PE, CCR7 PE, CD4 PECy7, and IFN-γ APC (all from eBioscience). Cells were resuspended in PBS supplemented with fetal bovine serum (FBS) (HyClone Thermo Scientific, Logan, UT, USA), stained with specific antibodies, fixed with IC fixation buffer (eBioscience) and resuspended in FACSFlow buffer (Becton Dickinson, San Diego, CA, USA) for subsequent analysis. Data were acquired on a FACSAria III with FACSDiva v6.1.3 software (both Becton Dickinson) and analyzed by FlowJo software (Treestar, USA).
9
Flow Cytometric Analysis of Liver NPCs
10
Comprehensive Flow Cytometry Phenotyping
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For flow cytometric analysis, cells were fixed in fixation/permeabilization buffer (eBioscience) overnight, washed and blocked in PBS containing 1% bovine serum albumin (BSA) and rat immunoglobulin (1µg/ml, Sigma, St. Louis, USA). Cells were stained with F4/80 APC or PerCP-Cy5.5, CD11b APC or FITC, Gr1 PE-Cy7, CD80 FITC, CD86 PE or APC, MHC-II PE or FITC, CD40 PE, Ly6C PerCP-Cy5.5 (all eBioscience) and SiglecF PE (BD Biosciences). To stain for RELMα-positivity, cells were pre-incubated in permeabilization buffer (eBioscience) and then stained with anti-RELMα (Peprotech, New Jersey, USA). Subsequently, cells were washed twice in permeabilization buffer and a secondary antibody (goat anti-rabbit Alexa488, Invitrogen, Carlsbad, USA) was used. As a control unspecific and isotope identical Alexa488 antibody (Invitrogen) was used. Data was acquired using a BD FACS Canto and BD FACSDiva software; for generation of figures and plots FlowJo software (Tree Star, Ashland, USA) was used.
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