Tissue cryosections were fixed in ice-cold acetone, washed, and then blocked with avidin/biotin agent (Vector Laboratories, Burlingame, CA, USA). To analyze the location and abundance of macrophages and neutrophils, cecal slides were stained with FITC-labeled anti-mouse F4/80 (eBioscience Cat. #11-4801-81) and Cy5-labeled anti-mouse Ly6G (Biolegend Cat. #108410). DNA was stained and mounted using the 4′, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). To examine the bacterial killing capacity, peritoneal macrophages were collected from uninfected WT and p40phox KO mice, grown on cover slips, and infected with S. Typhimurium for 1 h (cell/bacteria, 1:10). Cells were then stained with phycoerythrin (PE)-conjugated anti-F4/80 at both 2 and 6 h after infection (24 (link)). Internalization and elimination of bacteria by macrophages were visualized by detecting Salmonella with a rabbit antibody against Salmonella, followed by FITC-labeled anti-rabbit IgG antibody.
Fitc labeled anti mouse f4 80
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FITC-labeled anti-mouse F4/80 is a fluorescently-conjugated antibody that binds specifically to the F4/80 antigen expressed on the surface of mouse macrophages. It can be used to identify and quantify macrophage populations in various mouse samples using flow cytometry or microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin agent, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsr 2, by BD (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Rat anti mouse f4 80, by Abcam (1 mentions)
Dulbecco s modified eagle s medium dmem, by Wisent (1 mentions)
Tnf α enzyme linked immunosorbent assay elisa kit, by Cusabio (1 mentions)
Sds polyacrylamide gel electrophoresis, by Beyotime (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
4 protocols using fitc labeled anti mouse f4 80
1
Macrophage-Mediated Bacterial Clearance
2
Phagocytosis of Dead Cell Debris
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Peritoneal cells (106 cells/sample) derived from zymosan model mice on day 7 were mixed with dead cell debris labeled with FVD780 with or without AIM coating, in serum free DMEM/F-12 medium supplemented with 5 μg/ml insulin, 5 μg/ml transferrin and 5 ng/ml selenous acid at 37 °C for 10 and 30 min. Then, the cells were washed twice with ice-cold MACS buffer. Thereafter, the cells were incubated with Allophycocyanin (APC) labeled anti-mouse Mac-1(CD11b) antibody (eBioscience), Phycoerythrin (PE)-labeled anti-mouse Ly6G (Gr-1, eBioscience) and fluorescein isothiocyanate (FITC)-labeled anti-mouse F4/80 (eBioscience), and were re-suspended with MACS buffer containing 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The cells were then subjected to flow cytometry (BD LSRII, BD Biosciences, San Jose, CA). The proportion of engulfment of FVD780-positive cell debris within DAPI−Mac-1+F4/80+Gr-1− cells and DAPI−Mac-1+F4/80−Gr-1+ cells was assessed.
3
Flow Cytometric Characterization of Myeloid Cells
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Cell pellets from the air-pouch exudates were resuspended in PBS containing 1% BSA to block Fc-receptors for 30 min at 37°C and incubated with fluorescein isothiocyanate (FITC)-labeled anti-mouse F4/80, APC-labeled anti-mouse CCR7, and PE-labeled anti-mouse CD206 antibodies (eBioscience) for 1 h at room temperature in the dark. Corresponding isotype controls were also established. Finally, cells were analyzed using the methods described above.
4
Macrophage Phenotype Modulation in Vitro
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The following reagents were used in the study: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Wisent (Montreal, QC, Canada). Puromycin (PM), penicillin, and streptomycin were purchased from Sigma (St. Louis, MO, USA). SDS-polyacrylamide gel electrophoresis and 10% buffered neutral formaldehyde were purchased from Beyotime (Shanghai, China). Nitrocellulose membranes were provided by Millipore (Billerica, MA, USA). FITC-labeled anti-mouse F4/80, PE-conjugated anti-mouse programmed death ligand 1 (PD-L1), and PE-Cy5-labeled anti-mouse CD80 monoclonal antibodies were obtained from eBioscience (San Diego CA, USA) for flow cytometry analysis. Antibodies against MMP-2, MMP-9, and GAPDH used for Western blotting were manufactured by Proteintech (Chicago, IL, USA). Rat anti-mouse F4/80, CD68, IL-10, TGF-β, VEGF, and anti-mouse major histocompatibility complex class II (MHCII) monoclonal antibodies that recognize cells from both BALB/c and C57 BL/6 mice were manufactured by Abcam (Cambridge, MA, USA) for immunohistochemistry assays. The TNF-α enzyme-linked immunosorbent assay (ELISA) kit was provided by CUSABIO (Wuhan, China). The interleukin-6 (IL-6) ELISA kit was obtained from RayBiotech (Norcross GA, USA). The Greiss Reagent System determined for nitrite was purchased from Promega Biotech Company (Madison, WI, USA).
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