Tissue from the spleen tumour was minced and washed repeatedly with PBS. The resulting slurry was subjected to partial enzyme digestion (DNAse, Roche, Cat. no. 10104159001, Basel, Switzerland; Pronase, Roche, Cat. no. 10165921001, Basel, Switzerland; Collagenase, Gibco BRL, Cat. no. 17100, Schwalbach, Germany), passed through a 100 μm nylon mesh, and spun on a density gradient (Ficoll-Paque, Amersham Biosciences, Cat. no. 17544652, Piscataway, NJ). Cells at the interface were collected and washed twice in PBS. The resulting single-cell suspensions were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse/human CD11b (BioLegend, Cat. no. 101206, San Diego, CA), phycoerythrin (PE)-conjugated anti-mouse F4/80 (BioLegend, Cat. no. 123110, San Diego, CA) and Mouse MDSC Flow Cocktail 2 with Isotype Ctrl (BioLegend, Cat. no. 147003, San Diego, CA). Antibodies were diluted 1:100 in flow cytometric buffer (PBS with 5% FBS) and incubated with cells for 1 h at 4 °C. Cells were then washed and resuspended in antibody-free flow cytometric buffer. Flow cytometric measurement of fluorescence emission was performed on a total of 10,000 cells per dissociated tumour tissue by a Beckman/Coulter Epics XL-MCL flow cytometer with EXPO32 software (Beckman, CA).
Pe conjugated anti mouse f4 80
Manufactured by BioLegend
Sourced in United States
The PE-conjugated anti-mouse F4/80 is a laboratory reagent used to detect and identify mouse F4/80 antigen-positive cells, such as monocytes and macrophages, through flow cytometry or other immunoassays. It consists of a phycoerythrin (PE) fluorescent dye conjugated to an antibody specific for the mouse F4/80 antigen.
Automatically generated - may contain errors
Lab products found in correlation
Percp conjugated anti mouse cd11b, by BioLegend (3 mentions)
1 2 dimyristoyl rac glycero 3 methoxypolyethylene glycol 2000 mpeg dmg, by NOF corporation (3 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti mouse human cd11b, by BioLegend (1 mentions)
Ficoll paque, by Cytiva (1 mentions)
Epics xl mcl, by Beckman Coulter (1 mentions)
Dnase, by Roche (1 mentions)
Pronase, by Roche (1 mentions)
Fitc conjugated anti mouse cd4, by BioLegend (1 mentions)
Fitc conjugated anti mouse ly6g, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Distearoylphosphatidylcholine dspc, by Merck Group (1 mentions)
Elisa kits for il 4 and tnf α, by BioLegend (1 mentions)
Anti gapdh antibody, by Proteintech (1 mentions)
Elisa kits for tnf α, by BioLegend (1 mentions)
Temed, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Zymosan a, by Merck Group (1 mentions)
Penicillin streptomycin, by Sartorius (1 mentions)
Trypan blue, by Sartorius (1 mentions)
7 protocols using pe conjugated anti mouse f4 80
1
Isolation and Characterization of Murine Myeloid-Derived Suppressor Cells from Tumor Tissue
2
Multiparametric Immune Cell Profiling
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For analysis of surface molecules associated with CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and macrophages, single cell suspensions from spleens and tumors were stained with the following fluorescein-conjugated antibodies: FITC-conjugated anti-mouse CD4, PerCP-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse Gr-1, Percp-conjugated anti-mouse CD11b and PE-conjugated anti-mouse F4/80 (all the above antibodies were from Biolegend, San Diego, CA, USA).
3
Immunoassay and Flow Cytometry Analysis of Inflammatory Mediators
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The indicated reagents were obtained as follows: ELISA kits for mouse TNF-α, IL-10, IL-6, and IL-12 (Biolegend), and for mouse CCL2 and CCL5 (R&D systems); FITC-conjugated anti-mouse Gr-1, FITC-conjugated anti-mouse Ly6G, PE-conjugated anti-mouse F4/80, PerCP-conjugated anti-mouse CD11b, PB-conjugated anti-mouse Ly6C and PE/Cy7-conjugated anti-mouse TLR4 (Biolegend); APC-conjugated anti-mouse Tim4 (Miltenyi); Alx647-conjugated anti-mouse IgG, goat anti-mouse arginase-1, and rabbit anti-IFN-β antibodies (Abcam); rabbit anti-mouse 12/15-lipoxygenase antibody (Cayman Chemical); goat anti-mouse CD11b, goat anti-mouse β-actin and goat anti-PR3 antibodies (Santa Cruz Biotechnology); anti-p-STAT-1 and anti-p-STAT3 (Cell Signalling); anti-goat and anti-rabbit horseradish peroxidase-conjugated antibodies (Jackson ImmunResearch laboratories). Recombinant Gal-1 was generously provided by the laboratories of GR and Dr. Lichtenstein (Universidad de Buenos Aires, Buenos Aires, Argentina; Ben-Gurion University, Be'er Sheva, Israel); monoclonal anti-Gal-1 IgG, isotype IgG and polyclonal rabbit anti-Gal-1 antibody were obtained from the laboratory of GR, and used as described (Toscano et al., 2007 (link); Stowell et al., 2008 (link); Rostoker et al., 2013 (link)).
4
Phenotypic Characterization of M1 Macrophages
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Flow cytometry was used to analyze the phenotypical changes in M1-mediated markers in RAW 264.7 macrophages. Cells were centrifuged at 1500 rpm for 5 min and washed once with ice-cold PBS, then incubated them with Alexa Fluor® 647 anti-mouse CD206 (Becton Dickinson and Company, Franklin, NJ, USA), FITC-conjugated anti-mouse CD16/32 (Becton Dickinson and Company, Franklin, NJ, USA), and PE-conjugated anti-mouse F4/80 (BioLegend, San Diego, CA, USA) at room temperature in the dark for 30 min. Flow cytometry was performed using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Quantitative analysis of phenotypic changes in macrophages was performed by FlowJo VX10 software (BD, Biosciences, San Jose, CA, USA).
5
Characterization of Macrophage Polarization
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Ovalbumin (OVA), cholesterol and distearoyl phosphatidylcholine (DSPC) were acquired from Sigma-Aldrich, Inc. (St. Louis, MO). Lipidoid (C12-200) was purchased from Xinjiahecheng Medical Chemistry Corporation (Hubei, China). 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (mPEG-DMG) was purchased from NOF Corporation (Tokyo, Japan). The Giemsa staining kit for BALF was purchased from Baso Biotechnology Corporation (Guangdong, China).
Antibodies against F4/80 and CD206 were purchased from Santa Cruz Biotechnology (CA, USA). Antibodies against MBD2 and Arginase-1 were purchased from Abcam (MA, USA). The anti-GAPDH antibody was purchased from Proteintech (Hubei, China), while anti-Ym1 antibody was ordered from Thermo Fisher Scientific (PA, USA). PE-conjugated anti-human CD14, PE-conjugated anti-mouse F4/80, and APC-conjugated anti-mouse CD11c antibodies, ELISA kits for IL-4 and TNF-α were purchased from BioLegend (CA, USA).
Antibodies against F4/80 and CD206 were purchased from Santa Cruz Biotechnology (CA, USA). Antibodies against MBD2 and Arginase-1 were purchased from Abcam (MA, USA). The anti-GAPDH antibody was purchased from Proteintech (Hubei, China), while anti-Ym1 antibody was ordered from Thermo Fisher Scientific (PA, USA). PE-conjugated anti-human CD14, PE-conjugated anti-mouse F4/80, and APC-conjugated anti-mouse CD11c antibodies, ELISA kits for IL-4 and TNF-α were purchased from BioLegend (CA, USA).
6
Neutrophil Elastase Inhibitor Silevestat Protocol
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The following reagents were purchased as detailed: acrylamide/bis-acrylamide, fibronectin, lipopolysaccharide (LPS) (from Escherichia coli, clone 055:B5), staurosporine, TEMED, Tween-20, the neutrophil elastase inhibitor silevestat, and zymosan A from Sigma-Aldrich; clodronate from Liposoma BV; anti-goat horseradish peroxidase-conjugated immunoglobulin G (IgG) and anti-rabbit horseradish peroxidase-conjugated IgG from Jackson Immuno Research Laboratories; WesternBright™ ECL from Advansta, fetal calf serum (FCS), L-glutamine, penicillin–streptomycin, RPMI 1640 and trypan blue from Biological Industries, Kibbutz Beit Haemek; goat anti-mouse CD11b (M-19) polyclonal IgG and rabbit anti-human Lf (H-65) polyclonal IgG from SantaCruz Biotechnology; FITC-conjugated anti-mouse Gr-1, PE-conjugated anti-mouse F4/80, PerCP-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CXCR4 (clone BL6-146508) and enzyme-linked immunosorbent assay (ELISA) kits for TNFα, IL-6, IL-12, and IL-10 from Biolegend; cycloheximide (CHX) from Cayman Chemical; PE selection kit was purchased from Stem Cell Technologies; and Protease Inhibitors Cocktail was purchased from Roche. FKD and FKE peptides were organically synthesized by GL Biochem (Shanghai) Ltd.
7
Isolation and Differentiation of Bone Marrow-Derived Macrophages
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BM cells were isolated from femurs and tibias of mice and resuspended to
1×106 cells/mL in 30% L929-conditioned DMEM medium
supplemented with 20% (v/v) FBS, 5×10–5 M
β-mercaptoethanol, 20 mM HEPES, 1 mM sodium pyruvate, and 1%
penicillin/streptomycin (Weischenfeldt and
Porse, 2008 ). Culture medium was replenished four days after culture.
On day 7, cells were harvested and the degree of BMDM differentiation was
measured by flow cytometry analyses after staining with PE conjugated anti-mouse
F4/80 (BioLegend, San Diego, CA, USA, cat. #123110) and antigen presenting cells
(APC) conjugated anti-CD11b (BioLegend, cat. #101212) antibodies. More than
90% of cells displayed F4/80+CD11b+phenotype.
1×106 cells/mL in 30% L929-conditioned DMEM medium
supplemented with 20% (v/v) FBS, 5×10–5 M
β-mercaptoethanol, 20 mM HEPES, 1 mM sodium pyruvate, and 1%
penicillin/streptomycin (
Porse, 2008
On day 7, cells were harvested and the degree of BMDM differentiation was
measured by flow cytometry analyses after staining with PE conjugated anti-mouse
F4/80 (BioLegend, San Diego, CA, USA, cat. #123110) and antigen presenting cells
(APC) conjugated anti-CD11b (BioLegend, cat. #101212) antibodies. More than
90% of cells displayed F4/80+CD11b+phenotype.
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