Cells were fixed in 4% paraformaldehyde for 20 min at 4°C. Next, cells were rinsed with 0.25% Triton X-100 in phosphate-buffered saline (PBS) and incubated with 70% alcohol for 5 min before being incubated with blocking buffer for 30 min at room temperature. For F4/80 staining, cells were incubated with F4/80 (565612, BD) overnight at 4°C and then counterstained with Hoechst 33342 (14533-100MG, Sigma-Aldrich). Lastly, cells were mounted on glass slides and examined using a Nikon microscope.
F4 80
Manufactured by BD
Sourced in United States, Germany
F4/80 is a transmembrane glycoprotein that is expressed on the surface of mature macrophages and certain other myeloid cell populations. It functions as an adhesion molecule and is commonly used as a marker for the identification and isolation of macrophages in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BD (71 mentions)
Siglec f, by BD (13 mentions)
Facscalibur, by BD (12 mentions)
Cd11b, by BioLegend (12 mentions)
Mhcii, by BD (10 mentions)
F4 80, by BioLegend (8 mentions)
Fc block, by BD (8 mentions)
Ifn γ, by BD (8 mentions)
Lsrfortessa, by BD (8 mentions)
Cd11b, by Thermo Fisher Scientific (7 mentions)
Cd206, by BD (7 mentions)
Facsdiva software, by BD (7 mentions)
Cd62l, by BD (6 mentions)
Cd206, by BioLegend (6 mentions)
Facscanto 2, by BD (5 mentions)
Pd l1, by BD (5 mentions)
Tnf α, by BD (5 mentions)
Ionomycin, by Merck Group (5 mentions)
Cd16 32, by BD (5 mentions)
Facsaria 2, by BD (5 mentions)
149 protocols using f4 80
1
Immunostaining of Macrophages in Fixed Cells
2
Phenotypic Analysis of Immune Cells
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After the treatments, the expression of surface molecules on DC or Mϕ was quantified by flow cytometry using FITC- or PE-conjugated antibodies (CD11c, F4/80, I-Ad, I-Ab, CD40), all purchased from BD Bioscience. Peritoneal cell populations were studied by flow cytometry using F4/80, CD11c, CD3, and CD19 antibodies (BD Bioscience). Samples were collected using a flow FACSCanto II (BD Bioscience, San Jose, CA, USA), and data were analyzed by Flowing Software2 (Turku Centre for Biotechnology, University of Turku, Finland).
3
Peritoneal Exudate Cell Analysis in Sepsis
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Peritoneal lavage was harvested before and after cecal slurry peritonitis-induced polymicrobial sepsis. Peritoneal exudate cells were incubated with FITC, PE, PerCP, or APC-conjugated anti-CD11b (BD Pharmingen, San Diego, CA), anti-CD11c (BioLegend, San Diego, CA), anti-F4/80 (BD Pharmingen), and anti-Gr1 (BioLegend) monoclonal antibodies. Subpopulations of PMNs (CD11b+F4/80−Gr1hi) and macrophages (CD11b+F4/80+CD11clo) in the peritoneal cavity were analyzed by flow cytometry (BD Biosciences).
4
Tumor-associated Myeloid Subset Isolation
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Single cell suspensions were made from the lung tissues of tumor-bearing mice as described [50 (link)]. Gr-1+CD11b+, Ly6G+CD11b+Ly6C-F4/80-, Ly6C+CD11b+Ly6G-F4/80-, and F4/80+CD11b+ Ly6G-Ly6C- myeloid subsets were sorted from splenocytes, tumor tissues, and lungs as by FACSAria flow cytometer (BD). Antibodies including CD11b, Gr-1, Ly6G, Ly6C, F4/80, CD45, Lin, and CD31 were purchase from BD. CD117 was from eBioscience and Sca-1 was from Biolegend. FACS Calibur and Fortessa flow cytometer (BD, San Jose, CA) were used.
5
Epididymal Adipose Tissue SVC Isolation
6
Adipose Tissue Stromal Vascular Cell Isolation
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Epididymal visceral adipose tissue was mechanically chopped and then digested with collagenase Type 4, DNAse 1, and 0.5% fatty acid free bovine serum albumin in phosphate buffered saline (PBS) for 50 minutes at 37°C in an incubator shaker (120 RPM). Next, 0.5M EDTA was added with RPMI media and the cells were passed through a 70‐μm cell strainer rinsed with 2% fetal bovine serum (FBS) in PBS. The cells were centrifuged at 1400 RPM for 5 minutes at 4°C, primary adipocytes were aspirated off from the top layer of the supernatant and the pellet containing the stromal vascular cells (SVC) was then incubated with red blood cell lysis buffer for 1 minute on ice. Two percent FBS‐PBS was added to the SVCs and centrifuged at 1400 RPM for 5 minutes at 4°C and then passed through a 40‐μm cell strainer. SVCs were stained with the following fluorophore‐tagged antibodies obtained from BioLegend (San Diego, CA): Zombie Aqua, CD45 (PerCP‐Cy5.5), CD11b (FITC), CD19 (APC‐Cy7), F4/80 (PE), and MHCII (BV421). The following SVC subsets were analyzed using a BD LSRII flow cytometer: CD45+CD11b‐CD19+ (B cells), CD45+CD11b+F4/80+MHCII+ (macrophages), CD45+CD11b+F4/80+MHCII‐ (macrophages). All data were analyzed in FlowJo and gates were drawn from fluorescence minus one controls.
7
Peritonitis Induction and Cell Analysis
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For peritonitis, mice were injected i.v., with 2.5 mg OM85 followed 5 h later by an i.p. injection of 350 μg alum (Pierce). 12–14 h after alum injection, mice were sacrificed and peritoneal cavities were washed with 6 ml PBS. PECs were counted and analyzed by flow cytometry using a combination of antibodies against CD11b (M1/70), Ly6C (AL-21), Ly6G (1A8) (BD Pharmingen), antiCD16/32 (93), CD11c (N418), F4/80 (BM8) (eBioscience). For cellular subtype analysis the following gating strategies were used: neutrophils (CD11b+, Ly6Ghigh, F4/80−), eosinophils (CD11b+, SSChigh) and macrophages (Ly6G−, SSClow, CD11b+, F4/80+), recruitment was analyzed on a FACSCanto (BD Bioscience) by using the FLOWJO software (Tree Star).
8
Peritoneal Immune Cell Profiling in CLP
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Peritoneal lavage was harvested from mice at different time points before and after CLP. Peritoneal exudate cells were incubated with anti-CD11b (BD Pharmingen), anti-CD11c (BioLegend), anti-F4/80 (BD Pharmingen), and anti-Gr1 (BioLegend) monoclonal antibodies (mAbs) conjugated with FITC, PE, PerCP, or APC. FITC-, PE-, PerCP-, and APC-conjugated anti-mouse isotype-matched mAbs (BD Pharmingen; BioLegend) were used as negative controls. Subpopulations of macrophages (CD11b+F4/80−CD11clo) and PMNs (CD11b+F4/80−Gr1hi) in the peritoneal cavity were detected by FACScan analysis (BD Biosciences).
9
Isolated Renal Cell Phenotyping and Culture
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Single cell suspensions of isolated renal tissue from Sham-operated or CLP-treated mice were stained with 7-AAD to detect viable cells and an antibody cocktail containing CD326, CD45, F4/80, and CD11b (all purchased from BD). Cell suspensions were sorted using a FACS Aria (BD) FACS sorter, resulting in CD45-/CD326+ epithelial cells and CD45+/F4/80+/CD11b+ MΦ. The gating strategy is given in Supplementary Figure S1 . Cells were then transferred to the cell culture and short-term cultured for 24 h using Dulbecco’s modified Eagle’s medium (Gibco, Dreieich, Germany) for TEC culture and RPMI-1640 medium for rMΦ, each supplemented with penicillin 100 U/mL (Sigma-Aldrich, Taufkirchen, Germany), streptomycin 100 mg/mL (Sigma-Aldrich), and 10% FCS (Capricorn Scientific, Ebersdorfergrund, Germany).
Supernatants were harvested for AAS and cellular lysates were used for RNA isolation. RNA isolation and transcription were performed using the RNeasy Micro Kit (Qiagen, 74004, Hilden, Germany) and Sensiscript RT Kit (Qiagen, 205211) according to the manufacturer’s kit protocols.
Supernatants were harvested for AAS and cellular lysates were used for RNA isolation. RNA isolation and transcription were performed using the RNeasy Micro Kit (Qiagen, 74004, Hilden, Germany) and Sensiscript RT Kit (Qiagen, 205211) according to the manufacturer’s kit protocols.
10
Hepatic Macrophage Isolation and Characterization
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At 8 weeks after S. japonicum infection, the mouse livers were perfused and digested using the collagenase method, followed by Percoll density gradient centrifugation to isolate hepatic mononuclear cells as described previously (50 (link)). The cells were stained with the following fluorescence-conjugated antibodies: anti-SiglecF (clone E50-2440; BD Biosciences), anti-CD11b (clone M1/70; BD Biosciences), anti-F4/80 (clone BM8; eBioscience), and anti–PD-1 (clone J43; eBioscience). The PD-1lo macrophages (SiglecF–CD11b+F4/80+PD-1lo) and PD-1hi macrophages (SiglecF–CD11b+F4/80+ PD-1hi) were analyzed and sorted using a FACSAria II cell sorter (BD Biosciences).
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