F4 80 apc clone bm8

Manufactured by Thermo Fisher Scientific

F4/80-APC (clone BM8) is a monoclonal antibody conjugated with Allophycocyanin (APC) fluorescent dye. It is used for the identification and enumeration of mouse F4/80-positive cells by flow cytometry.

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5 protocols using f4 80 apc clone bm8

Isolation and Immunophenotyping of Microglia

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Microglia cells were isolated by mechanical dissociation and Percoll gradient centrifugation as previously described (Verheijden et al., 2015 (link)). For immunological profiling, 1 × 10⁵ cells were incubated for 20 min at 4°C using combinations of CD11b-Pe Cy7 (clone M1/70, Biolegend) with either CD11c-APC (clone HL3, BD Pharmingen), F4/80-APC (clone BM8, eBioscience), CD204-Alexa 647 (clone MR5D3, AbD Serotec), CD206-Alexa 647 (clone 2F8, AbD Serotec) antibodies all at dilutions of 1:200 in a total volume of 200 μl. Flow cytometry was performed on a FACS Verse (BD Biosciences), and data was analyzed with BD FACsuite software (BD Biosciences). Injection and subsequent analysis of Bromodeoxyuridine (BrdU; FITC BrdU Flow Kit 559619, BD Biosciences) in mice by flow cytometric analysis has been described (Verheijden et al., 2015 (link)).

Beckers L., Stroobants S., D’Hooge R, & Baes M. (2018). Neuronal Dysfunction and Behavioral Abnormalities Are Evoked by Neural Cells and Aggravated by Inflammatory Microglia in Peroxisomal β-Oxidation Deficiency. Frontiers in Cellular Neuroscience, 12, 136.

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Comprehensive NPC Cell Phenotyping

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Purified NPC were treated with red blood cell lysis buffer, and cell preparations were stained with the following antibody cocktail: CD8a-Pacific Blue (clone 53-6-7, Thermo Fisher Scientific, Waltham, MA), CD4-PerCP Cy5.5 (clone RMA 4-5, eBioscience, San Diego, CA), CD11b-FITC (clone MI-70, eBioscience), NK1.1-PE-Cy7 (clone PKI 36, eBioscience), Tie2-PE (clone TEK4, eBioscience), F4/80-APC (clone BM8 eBioscience). Incubation was performed at 4°C for 20 minutes, followed by fixation at 37°C for 10 minutes in 4% formaldehyde. 20,000 cells were examined with a BD LRS Fortessa (BD Biosciences, San Jose, CA), and data analyzed with FlowJo software version 10. Live/Dead^® Yellow staining kit was used to confirm cell viability (Thermo Fisher Scientific). Two gates were designed using FSC-A vs. SSC-A plots (Figure 2C), as previously described ^{23 (link)}. G1 was further used to analyze lymphocytes T CD4+ (LT CD4+), CD8+ (LT CD8+) and natural killer (LT NK) using CD4, CD8a and NK1.1 markers. G2 was further sorted using Tie2 and CD11b markers, with CD11b^int/high Tie2^int/low subsequently used to analyze Kupffer and myeloid cells using CD11b and F4/80 markers.

Etienne-Mesmin L., Vijay-Kumar M., Gewirtz A.T, & Chassaing B. (2016). Hepatocyte Toll-Like Receptor 5 Promotes Bacterial Clearance and Protects Mice Against High-Fat Diet–Induced Liver Disease. Cellular and Molecular Gastroenterology and Hepatology, 2(5), 584-604.

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Comprehensive Immune Cell Profiling

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Red blood cells were lysed in bone marrow and collagenase IV (Sigma)-treated lung samples. Lung, spleen or BM cells (3 × 10⁶) were then stained with fixable viability dye eFluor501 (eBioscience) at the concentration of 1:1 000 for 30 minutes (4°C). Subsequently, the cells were washed with PBS supplemented with 0.5% BSA (Wisent) and incubated with anti-CD16/32 (clone 93, eBioscience) at a concentration of 1:100 in PBS/0.5% BSA at 4°C for 10. After washing, cells were incubated with fluorochrome tagged antibodies at 4°C for 30 minutes. Antibodies for the innate panel: anti-CD11b–Pacific Blue (clone M1/70, eBioscience), anti-CD11c–PE-Cy7 (clone HL3, BD Bioscience), Siglec-F–PE-CF594 (clone E50–2440, BD Bioscience), F4/80–APC (clone BM8, eBioscience), Ly6C–FITC (clone AL-21, BD Bioscience), Ly6G–PerCP-eFluor710 (clone 1A8, eBioscience). Antibodies for the adaptive panel: anti-CD3–PE (clone 145-2C11, eBioscience), anti-CD19–PE-Cy7 (clone eBio1D3 (1D3), eBioscience), anti-CD4–eFluor450 or anti-CD4-FITC (clone GK1.5, eBioscience), anti-CD8–AF700 (clone 53-6.7, BD Bioscience). All cells were subsequently washed with PBS/0.5% BSA and resuspended in 1% paraformaldehyde.

Khan N., Downey J., Sanz J., Kaufmann E., Blankenhaus B., Pacis A., Pernet E., Ahmed E., Cardoso S., Nijnik A., Mazer B., Sassetti C., Behr M.A., Soares M.P., Barreiro L.B, & Divangahi M. (2020). M. tuberculosis Reprograms Hematopoietic Stem Cells to Limit Myelopoiesis and Impair Trained Immunity. Cell, 183(3), 752-770.e22.

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Murine Myeloid Cell Immunophenotyping

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Peripheral blood was isolated from mice and RBCs were lysed with ammonium chloride solution (Stemcell Technologies). Cells were subsequently plated and blocked with anti-mouse FcR antibody (αCD16/CD32, BioLegend) in FACS buffer (PBS with 2% BSA and 1mM EDTA). Cells were then surface-stained with CD11b-BV421 (clone M1/70, BioLegend), F4/80-APC (clone BM8, eBioscience), Ly6G-FITC (clone 1A8, Miltenyi Biotec), CD115-PerCP-Cy5.5 (clone AFS98, Biolegend), and TLR2-PE-Vio770 (clone REA109, Miltenyi Biotec) according to manufacturer’s recommendations. Data were collected on the BD LSRII Flow Cytometer (BD Biosciences) platform and exported for analysis via FlowJo (v.9.0; Treestar, Inc, Ashland, OR).

Jablonski K., Young N.A., Henry C., Caution K., Kalyanasundaram A., Okafor I., Harb P., Schwarz E., Consiglio P., Cirimotich C.M., Bratasz A., Sarkar A., Amer A.O., Jarjour W.N, & Schlesinger N. (2020). Physical activity prevents acute inflammation in a gout model by downregulation of TLR2 on circulating neutrophils as well as inhibition of serum CXCL1 and is associated with decreased pain and inflammation in gout patients. PLoS ONE, 15(10), e0237520.

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Immune Cell Phenotyping in Liver Tissue

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Purified NPC were treated with red blood cell lysis buffer, and cell preparations were stained with the following antibody cocktail: CD8a-Pacific Blue (clone 53-6-7; Thermo Fisher Scientific, Waltham, MA), CD4-PerCP Cy5.5 (clone RMA 4-5; eBioscience, San Diego, CA), CD11b–fluorescein isothiocyanate (clone MI-70; eBioscience), NK1.1-PE-Cy7 (clone PKI 36; eBioscience), Tie2-PE (clone TEK4; eBioscience), and F4/80-APC (clone BM8; eBioscience). Incubation was performed at 4°C for 20 minutes, followed by fixation at 37°C for 10 minutes in 4% formaldehyde. A total of 20,000 cells were examined with a BD LRS Fortessa (BD Biosciences, San Jose, CA), and data were analyzed with FlowJo software version 10 (Ashland, OR). The Live/Dead Yellow staining kit was used to confirm cell viability (Thermo Fisher Scientific). Two gates were designed using forward scatter-A vs side scatter-A plots (Figure 2C), as previously described.²³ (link) G1 was used further to analyze lymphocytes T CD4+ (LT CD4+), CD8+ (LT CD8+), and natural killer (LT NK) using CD4, CD8a, and NK1.1 markers. G2 was sorted further using Tie2 and CD11b markers, with CD11b^int/high Tie2^int/low subsequently used to analyze Kupffer and myeloid cells using CD11b and F4/80 markers.

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