BM-MΦs and BM-DCs were stained with the following antibodies for phenotypic analysis: Fluorochrome-conjugated monoclonal antibodies against CD11b (M1/70; dilution 1:200), CD11c (N418; dilution 1:100), CD115 (AFS98; dilution 1:100), CD80 (16-10A1; dilution 1:100), CD86 (GL1; dilution 1:100), F4/80(BM8; dilution 1:50) and MHC class II (AF6-120.1; dilution 1:100) were from BD Biosciences Pharmingen. Monoclonal antibodies against TLR4 (Sa15-21 and MTS510) were from BioLegend. The Sa15-21 antibody was labelled with biotin (Sigma). Both anti-TLR4 antibodies were added at a dilution of 1:50. PerCp-Cy5.5-conjugated (dilution 1:50) and biotin-conjugated (dilution 1:100) monoclonal antibodies against CD14 (Sa2-8) were from eBioscience. Data were acquired using a FACSCalibur (Becton-Dickinson) and expressed as delta the mean fluorescent intensity (MFI of the sample−MFI of the isotype control).
F4 80 bm8
Manufactured by BD
F4/80 (BM8) is a monoclonal antibody that recognizes the F4/80 antigen, a glycoprotein expressed on the surface of mature mouse macrophages and microglia. It is a widely used marker for the identification and isolation of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cd11c n418, by BD (3 mentions)
Cd44 im7, by BD (3 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Ghost dye red 780, by Cytek Biosciences (2 mentions)
Fortessa, by BD (2 mentions)
Cd19 6d5, by BioLegend (2 mentions)
I a 1 e m5 114, by BioLegend (2 mentions)
Cd4 gk1, by BD (2 mentions)
Cd8 53 6, by BD (2 mentions)
Cd11b m1 70, by BD (2 mentions)
T bet 4b10, by BioLegend (2 mentions)
Cd11c n418, by BioLegend (2 mentions)
Cd45.1 a20, by BioLegend (2 mentions)
Cd3e 145 2c11, by BD (2 mentions)
Siglecf e50 2440, by BD (2 mentions)
Facscalibur, by BD (1 mentions)
Cd86 gl1, by BD (1 mentions)
Cd80 16 10a1, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
8 protocols using f4 80 bm8
1
Phenotypic analysis of BM-MΦs and BM-DCs
2
Multiparametric Flow Cytometry Analysis
3
Multiparametric Flow Cytometry Analysis of MLN Cells
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Flow cytometry analysis was performed according to the methods described previously [28 (link)]. Briefly, the cells isolated from the MLN (the MLN cells) were incubated with an Fcγ receptor-blocking mAb (CD16/32; 2.4G2, BD Biosciences) for 15 minutes at 4°C. For surface antigen detection, the cells were labeled with monoclonal antibodies against Gr-1 (RB6-8C5, BD Biosciences), F4/80 (BM8, BD Biosciences), αβTCR (H57-597, BD BioLegend), γδTCR (GL3, BioLegend), NK1.1 (PK136, BioLegend), CD4 (RM4-5, BD Biosciences), CD44 (IM7, BD Biosciences), CD25 (PC61, BioLegend), B220 (RA3-6B2, BioLegend), and CD19 (MB19-1, BioLegend) for 30 min at 4°C.
For intracellular cytokine staining, the cells isolated from the MLN (the MLN cells) were stimulated with ionomycin (1 mg/mL; Sigma-Aldrich) and PMA (25 ng/mL; Sigma-Aldrich) for 5 h at 37°C, with brefeldin A (10 mg/mL; Sigma-Aldrich) added after 1 h. Then, the cells were fixed and permeabilized with fixation/permeabilization working solution for 20 min at 4°C followed by incubation with monoclonal antibodies against IFN-γ (XMG1.2, BD Biosciences), IL-17A (TC11-18H10.1, BD Biosciences), and IL-10 (JES5-16E3, BD Biosciences). The cells were analyzed using a Cytofix/Cytoperm Plus Kit.
For intracellular cytokine staining, the cells isolated from the MLN (the MLN cells) were stimulated with ionomycin (1 mg/mL; Sigma-Aldrich) and PMA (25 ng/mL; Sigma-Aldrich) for 5 h at 37°C, with brefeldin A (10 mg/mL; Sigma-Aldrich) added after 1 h. Then, the cells were fixed and permeabilized with fixation/permeabilization working solution for 20 min at 4°C followed by incubation with monoclonal antibodies against IFN-γ (XMG1.2, BD Biosciences), IL-17A (TC11-18H10.1, BD Biosciences), and IL-10 (JES5-16E3, BD Biosciences). The cells were analyzed using a Cytofix/Cytoperm Plus Kit.
4
Multiparametric Flow Cytometry Analysis
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Tetramer-enriched samples were stained for surface markers for 30 minutes on ice using antibodies to: CD4 (GK1.5, BD), CD8 (53–6.7, BD), CD90.1 (HIS51), CD90.2 (53–2.1, 30-H12), CD3e (145-2C11, BD), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD44 (IM7, BD), CD45.1 (A20, Biolegend), CD45.2 (104), B220 (RA3–6B2), and/or PD1 (J43). Cellular viability was confirmed using GhostDye Red 780 (Tonbo Biosciences). For transcription factor expression analysis, stained cells were fixed and permeablized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Cells were stained overnight at 4°C with antibodies to T-bet (4B10, Biolegend), Foxp3 (FJK-16s), RORγt (Q31-378, BD), and Bcl-6 (K112-91, BD). For thymic dendritic cell and thymic epithelial cell analyses cells, following enrichment cells were stained with antibodies to CD11c (N418), CD19 (6D5, Biolegend), I-A/I-E (M5/114.15.2, Biolegend), CD8 (53–6.7, BD), CD90.2 (53–2.1, 30-H12), CD11b (M1/70), and CD45.2 (104, BD). Antibodies were purchased from eBioscience unless otherwise indicated. Cells were analyzed by flow cytometry on a Fortessa (BD). Data were analyzed using FlowJo software (TreeStar).
5
Immunophenotyping Flow Cytometry Protocol
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Annexin V and 7-AAD were obtained from BD (Franklin Lakes, NJ) and Live/Dead Aqua from Life Technologies Corporation (Eugene, OR). Antibodies used in the study were the following: Fc block/anti-CD16/32 (2.4G2) (BD) and directly conjugated monoclonal antibodies (mAb/clone): CD45.2/104 (Invitrogen, Grand Island, NY), SiglecF/E50-2440, Ly6G/1A8, Ly6C/HK1.4, Axl/175128 (all from BD), F4/80/BM8, MHCII/M5-114.15.2, CD206/C068C2, MerTk/DS5MMER (all from Biolegend, San Diego, CA), CD11c/N418 and CD11b/M1-70 (both from eBioscience, San Diego, CA).
6
Tumor Immune Cell Phenotyping
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The anti-mouse CD16/32 antibody (clone
number 93) was added to single-cell suspensions from primary tumors
and lungs to block nonspecific binding of immunoglobulin to macrophage
Fc receptors, followed by staining with the following antibodies:
anti-mouse CD45 (30-F11), CD11b (M1/70, BD Biosciences), and F4/80
(BM8). All antibodies were obtained from eBioscience, unless otherwise
indicated. An additional antibody used for flow cytometry analysis
included CD206 (Abcam), which was followed by staining with the appropriate
secondary antibodies. Data were acquired using LSRFortessa (BD Bioscience)
and analyzed using the FlowJo software (Tree Star).
number 93) was added to single-cell suspensions from primary tumors
and lungs to block nonspecific binding of immunoglobulin to macrophage
Fc receptors, followed by staining with the following antibodies:
anti-mouse CD45 (30-F11), CD11b (M1/70, BD Biosciences), and F4/80
(BM8). All antibodies were obtained from eBioscience, unless otherwise
indicated. An additional antibody used for flow cytometry analysis
included CD206 (Abcam), which was followed by staining with the appropriate
secondary antibodies. Data were acquired using LSRFortessa (BD Bioscience)
and analyzed using the FlowJo software (Tree Star).
7
Synthesis and Validation of Maresin Analogs
8
Comprehensive Immune Cell Profiling
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Cell-surface markers were assessed by staining cells. Blocking was performed with purified anti-mouse CD16/32 (Fc block, BD Biosciences), and the samples were incubated for 30 min at 4 °C with fluorochrome-conjugated antibodies against CD45 (30-F11), CD3 (17A2), CD11b (M1/70), Ly6C (HK1.4), CD11c (N418), MHCⅡ (M5/114.15.2), Ly6G (1A8), CD103 (2.00E+07) and CD206 (C068C2) at a 1:400 dilution and F4/80 (BM8) at a 1:100 dilution. These antibodies were purchased from BioLegend. Monitoring of apoptosis was performed by staining with anti-Annexin V antibody and PI (BD Biosciences). Cells negative for PI staining but positive for anti-Annexin V staining were defined as early apoptotic cells, and those double positive for PI and anti-Annexin V staining were defined as late apoptotic cells. Stained cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences) and the data were processed using the FlowJo software (Tree Star).
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