The primary microglia and astrocytes were incubated with F4_80-PE (1:500, 565410; BD Biosciences, Franklin Lakes, NJ, USA) or glial fibrillary acidic protein (GFAP)-PE (1:500, 561483; BD Biosciences) for 30 min at 4 °C. For polarization analysis, treated microglia were collected and added to F4_80-PE (565410, BD Biosciences), iNOS-FITC (1:500, 610330; BD Biosciences), and CD206-APC (1:500, 17-2061-82; Thermo Fisher Scientific, Waltham, MA, USA) for incubation. Microglia were loaded in a flow cytometer (FACSVerse 8, BD) and the polarization was visualized. The obtained data were analyzed using FlowJo software (Version 7.6.1; FlowJo, Treestar, OR, USA).
F4 80 pe
Manufactured by BD
Sourced in United States
F4/80-PE is a laboratory reagent used in flow cytometry applications. It is a fluorescently-labeled antibody that binds specifically to the F4/80 antigen, which is expressed on the surface of macrophages and other myeloid cells. The PE (Phycoerythrin) fluorescent label allows for the detection and quantification of F4/80-positive cells in a sample.
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Lab products found in correlation
Cd11b fitc, by BD (10 mentions)
Facscalibur, by BD (7 mentions)
Cd11b percp, by BD (5 mentions)
Cd45 apc cy7, by BD (5 mentions)
Cd11b pe cy7, by BD (5 mentions)
Cd11b pe, by BD (5 mentions)
Ly6c fitc, by BD (4 mentions)
Cd11b bv421, by BD (4 mentions)
Cd8 apc, by BD (4 mentions)
Cd11b apc, by BD (4 mentions)
Cd11c fitc, by BD (4 mentions)
Cd4 fitc, by BD (4 mentions)
Ly6g fitc, by BD (4 mentions)
Software, by FlowJo (3 mentions)
Ly6c apc, by BD (3 mentions)
Cd4 apc, by BD (3 mentions)
Cd3 fitc, by BD (3 mentions)
Cd45 percp, by BD (3 mentions)
Cd19 pe, by BD (3 mentions)
Cd8 fitc, by BD (3 mentions)
60 protocols using f4 80 pe
1
Microglia and astrocyte polarization analysis
2
Quantifying Neuronal Apoptosis and Microglial Activation
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The degree of neuronal apoptosis was detected using the Apoptosis Detection Kit (YiFeiXue Biotechnology) according to manufacturer’s instructions. Neurons were incubated with Annexin V-FITC reagent at room temperature for 10 min, followed by incubation with Propidium Iodide reagent for 5 min. Primary microglia were incubated with F4_80-PE (565,410, BD Biosciences, Franklin Lakes, NJ, USA) and iNOS-FITC (610,330, BD Biosciences) for 30 min at 4 °C, injected into a flow cytometer (FACSVerse 8, BD) and the obtained data were analyzed using FlowJo software (Version 7.6.1; FlowJo, Treestar, OR, USA).
3
Isolation and Analysis of Mouse Immune Cells
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The tissues were cut and homogenized in a homogenizing apparatus and 10 ml sterile saline was added to prepare a cell suspension that was filtered through a 200-micron nylon mesh. The cell suspension was centrifuged at 1500 × g for 5 min, and then the supernatant was discarded. The precipitate was resuspended by adding red blood cell lysate and was allowed to stand at room temperature for 5 min. After neutralization of the cell suspension with PBS, centrifugation was performed at 1500 × g for another 5 min, and then the supernatant was discarded. The cell suspension was then washed and resuspended once again with PBS and then incubated with the rat anti-mouse CD4-FITC, F4/80-PE, Ly6G and LY-6C-FITC (BD Biosciences, United States) for 30 min on ice. Finally, the cells were analyzed by flow cytometry (BD Biosciences, United States).
4
Isolation and Flow Cytometry Analysis of Aortic Cells
5
Immunophenotyping of Mouse Blood Cells
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The heparinized blood from each animal was divided into two parts. A volume of 150 µl was designated for blood count and measured using an ABX Pentra 60 C+ hemoanalyzer (Horiba, Kyoto, Japan). The rest of the blood (200–300 µl) was lysed using EasyLyse™ (Dako, Glostrup, Denmark). The amount of cells in suspension was as determined by Turk’s solution (2% acetic acid; Sigma-Aldrich) using a hemocytometer chamber. Cells (5 × 105/100 µl) were marked with two panels of monoclonal antibodies. The first panel was delineated to detect T, B, and NK lymphocytes (CD3ϵ, CD4, CD8, CD19, and NK1.1). The second panel was designed for determination of monocytes and neutrophils (CD11b, F4/80, Ly6C, and Ly6G). The following monoclonal antibodies with fluorochromes for flow cytometry analysis were purchased from BioLegend (San Diego, CA, United States): anti-mouse CD3ϵ–FITC, CD4–BV421, CD19–PE, CD11c–BV421, CD45–APC/Fire™750, F4/80–PE, and from BD Bioscience: CD8–PECy7, CD11b–BV510, Ly6C–FITC, Ly6G–PECy7, and NK1.1–APC.
6
Murine Dendritic Cell Characterization
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Female C57BL/6 mice purchased from Ji'nan Pengyue Laboratory Animal Breeding Co., Ltd. (China) were housed and maintained in the animal facilities of Weifang Medical University. Recombinant murine GM-CSF and IL-4 were purchased from R&D Systems (USA). A Fluo-4 Direct™ calcium assay kit was purchased from ThermoFisher Scientific (USA). ARL-67156, Bz-ATP, OX-ATP, and suramin were purchased from Tocris Bioscience (USA). A-438079, a competitive P2X7 receptor antagonist, was purchased from Santa Cruz (USA). Luminescent ATP Detection Assay kit was purchased from Abcam (USA). The endocytosis inhibitors MDC, PitS2, Dynasore, and P2X4-specific antagonist PSB-12062 were purchased from Sigma-Aldrich (USA). RNAiso Plus, SYBR Premix Ex Taq II, and the PrimeScriptTM RT reagent Kit were obtained from Takara (China). CD11c-APC (#550261), CD11b-FITC (#553310), MHC II-PE (#558593), and F4/80-PE (#565410) and isotype control antibodies were purchased from BD Bioscience (USA). CD39-PE (#12-0391-82) was purchased from Thermo Fisher (USA). All experiments involving animals were performed in accordance with the Chinese National Laboratory Animal-Guideline for Ethical Review of Animal Welfare and approved by the Institutional Animal Care and Use Committee of Weifang Medical University (NO. 010/2017).
7
Murine Macrophage Activation Assay
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Sphingosine, sphinganine, S1P, dhS1P, P1P, OVA grade V and LPS were procured from Sigma-Aldrich (St. Louis, MO, USA). Murine recombinant IFN-γ was obtained from PeproTech (London, UK). Antibody against S1PR4 and mouse IL-1β, IL-6, and IL-12b ELISA kit were procured from Boster (Wuhan, Hubei, China). Fluorescent antibody against S1PR4 was obtained from Affinity Biosciences (Cincinnati, OH, USA). Antibodies against iNOS and formyl peptide receptor 2 (FPR2) were purchased from Abcam (Cambridge, MA, USA). Antibodies against p38, p-p38, p65, p-p65, JNK, P-JNK, Erk, p-Erk, FLAG and magnetic beads were procured from Cell Signaling Technology (Danvers, MA, USA). Antibodies against CD68 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Arg-1, IL-1β, and β‐Actin were procured from the Proteintech Group (Wuhan, Hubei, China). Anti-mouse CD45-APC/Cy7, F4/80-PE, CD11b-V450, and CD86-FITC were procured from BD Biosciences (San Jose, CA, USA). Anti-mouse CD206-PE/Cy7 and murine recombinant IL-4 were procured from BioLegend (San Diego, CA, USA). CYM50358 was obtained from Tocris Bioscience (Bristol, UK). All other reagents were obtained from MedChem Express (Monmouth Junction, NJ, USA), except stated otherwise.
8
Murine Immune Cell Profiling by Flow Cytometry
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Peripheral blood samples from mice were collected in anticoagulant tubes. Erythrocytes were lysed with lysis buffer (BD Biosciences, Franklin Lakes, NJ, USA), and the remaining cells were washed with 2 ml PBS. Spleen and lymph nodes were mechanically disrupted and passed through a 70-μm membrane filter to obtain single-cell suspensions that were washed with RPMI-1640 medium and labeled with the following antibodies: CD45-V500, CD3-allophycocyanin (APC)-Cy7, CD4-fluorescein isothiocyanate, CD8-peridinin chlorophyll protein-Cy5.5, NK1.1-BV650, CD11b-BV421, GR1-BV605, Ly6G-phycoerythrin (PE)-Cy7, Ly6C-APC, and F4/80-PE (BD Biosciences). Cells were sorted by flow cytometry (LSRFortessa; BD Biosciences), and the data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
9
Immunophenotyping of Murine Macrophages
10
Isolation and Expansion of Mouse Bone Marrow-Derived Macrophages
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Mouse bone marrow-derived macrophage (BMMØ) isolation and ex vivo expansion was performed as previously described with slight modifications [14 (link)]. Femur and tibial bone marrow cavities were flushed, resuspended in the DMEM complete medium [(DMEM supplemented with 10% (vol/vol) FBS, 1% (vol/vol) streptomycin/penicillin and 50% (vol/vol) and L929 conditioned medium (as a macrophage colony-stimulating factor (M-CSF))] was passed through a 70-μm-cell strainer and then incubated in a humidified chamber at 37 °C with 5% CO2. After differentiation for 5–6 days, cells were subcultured (passage 1), maintained, and used for flow cytometry analysis using CDD11b-APC (1:100; #553312; BD Biosciences) and F4/80-PE (1:100; #565410; BD Biosciences).
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