Anti f4 80 clone bm8

Manufactured by BioLegend

Sourced in United States

Anti-F4/80 (clone BM8) is a mouse monoclonal antibody that recognizes the F4/80 antigen, a cell surface glycoprotein expressed on murine macrophages.

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Lab products found in correlation

Anti cd11b clone m1 70, by BioLegend (8 mentions) Anti ly6g clone 1a8, by BioLegend (6 mentions) Anti cd11b clone m1 70, by BD (6 mentions) Anti cd45 clone 30 f11, by BioLegend (5 mentions) Anti cd19 clone 6d5, by BioLegend (5 mentions) Anti cd3 clone 17a2, by BioLegend (5 mentions) Anti ly6c clone hk1, by BioLegend (5 mentions) Anti cd11c clone n418, by BioLegend (5 mentions) Anti cd11c clone hl3, by BD (4 mentions) Facscanto 2 flow cytometer, by BD (3 mentions) Anti cd62l clone mel 14, by BioLegend (3 mentions) Anti iaie clone m5 114, by BioLegend (3 mentions) Anti gr 1 clone rb6 8c5, by BioLegend (3 mentions) Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (3 mentions) Lsrfortessa, by BD (3 mentions) Facscalibur, by BD (3 mentions) Anti cd11c hl3, by BD (2 mentions) Mr5d3, by OriGene (2 mentions) Anti tnf mp6 xt22, by BioLegend (2 mentions) Anti inos cxnft, by Thermo Fisher Scientific (2 mentions)

26 protocols using anti f4 80 clone bm8

Epididymal Fat Pad Characterization

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Seven and 14 days after implant, epididymal fat pads were harvested following euthanasia and washed in ice cold PBS (Sigma). Fat pads were minced, digested in collagenase (Liberase TL, Roche), and passed through a 100 μm filter. The stromal vascular fraction was harvested by centrifugation, washed in MACS buffer (PBS, 0.5 mM EDTA, 30% BSA), and incubated with anti-CD16/32 prior to adding an antibody cocktail against extracellular antigens. The following antibodies were purchased from Biolegend: anti-CD45 clone 30-F11, anti-Ly6G clone 1A8, anti-F4/80 clone BM8, anti-NK1.1 clone PK136, anti-CD19 clone 6D5, anti-CD11b clone M1/70, anti-CD3 clone 17A2, and Trustain fcX (anti-CD16/32) clone 93. The following isotype controls were also purchased from Biolegend: mouse IgG2a clone MOPC-173; rat IgG2a clone RTK2758; rat IgG2b clone RTK4530. After antibody incubation, cells were washed, fixed, and analyzed using a FACS Aria flow cytometer (BD Biosciences). The number of CD45 cells in each flow cytometry sample was calculated using Bang’s labs Flow Cytometry Absolute Count Standard, which was added prior to data acquisition. FlowJo software (Treestar) was utilized to compensate and analyze data. FMOs with isotype controls were used to determine specific antibody signal. The gating scheme used in the flow cytometry analysis is depicted in Figure S3.

Murphy K.P., Hendley M.A., Isely C., Annamalai P., Peña E, & Gower R.M. (2018). Resveratrol Delivery from Porous Poly(lactide-co-glycolide) Scaffolds Promotes an Anti-Inflammatory Environment within Visceral Adipose Tissue. ACS applied materials & interfaces, 10(50), 43363-43374.

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Multicolor Flow Cytometry Immunophenotyping

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Anti-gp38 (clone 8.1.1), anti-CD31 (clone 390), anti-CD11b (clone M1-70), anti-CD11c (clone N418), anti-CD45.1 (A20), anti-IAb (AF6.120.1), anti-CD80 (clone 16-10A1), anti-CD86 (clone GL1), anti-PD-L1 (clone 10F.9G2), anti-CD63 (clone NVG-2), and anti-F4/80 (clone BM8) mAbs were from BioLegend. Anti-CD45 (clone 30F11), anti-CD103 (clone M290), anti-CD16/32 FcyRIII (clone 2.4G2), anti-I-A^d/I-E^d (clone 2G9), anti–Annexin V mAbs were from BD. Anti-CD19 (1D3), anti-CD8 (clone 53–6.7), anti-CD40 (clone HM40-3), and anti-CD4 (GK1.5) were from eBioscience. Anti-CD3 and anti-CD28 were from Bio X Cell. For flow cytometry, enriched CD45^neg cells were stained with mAbs against CD45, gp38, and CD31. In certain experiments, cells were further stained with mAbs against MHCII or isotype control as indicated. Cells were either acquired on a FACSCalibur (BD) or sorted using a MoFlowAstrios (Beckman Coulter), and analyzed using FlowJo software (Tree Star).

Dubrot J., Duraes F.V., Potin L., Capotosti F., Brighouse D., Suter T., LeibundGut-Landmann S., Garbi N., Reith W., Swartz M.A, & Hugues S. (2014). Lymph node stromal cells acquire peptide–MHCII complexes from dendritic cells and induce antigen-specific CD4+ T cell tolerance. The Journal of Experimental Medicine, 211(6), 1153-1166.

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Isolation and Characterization of Stromal Vascular Fraction

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SVF was prepared as reported55 (link),59 (link),60 (link). Briefly, inguinal SAT or VAT were isolated from euthanized mice, minced and digested for 60 min in DMEM containing collagenase type I (2 mg/ml per gram of tissue; Invitrogen) at 37°C. Cell suspensions were then filtered through a 40-μm cell strainer and the SVF was collected as a pellet after centrifugation at 500g for 5 min. After RBC lysis, cells were stained with anti-CD45 (30-F11, BD), anti-F4/80 (clone BM8, BioLegend), anti-CD11b (M1/70, BD), anti-CD11c (HL3, BD), anti-CD206 (c06802, BioLegend; MR5D3, Acris), anti-TNF (MP6-XT22, BioLegend), anti-iNOS (CXNFT, eBioscience) and anti-CD49d [clone PS/2, AbD Serotec or clone 9C10(MFR4.B), BioLegend]. Cells were analyzed in a FACS Canto II flow cytometer (BD).

Chung K.J., Chatzigeorgiou A., Economopoulou M., Garcia-Martin R., Alexaki V.I., Mitroulis I., Nati M., Gebler J., Ziemssen T., Goelz S.E., Phieler J., Lim J.H., Karalis K.P., Papayannopoulou T., Blüher M., Hajishengallis G, & Chavakis T. (2017). A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity. Nature immunology, 18(6), 654-664.

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Characterization of Hematopoietic Stem Cells in Fbn1 Mutant Mice

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Marrow stromal cells were flushed out from femur and tibia of WT and Fbn1^Prx1−/− mice. For LSK-HSC analyses, approximately 10⁶ bone marrow cells were labeled with antibodies anti-Lineage cocktail (APC), anti-CD34-FITC (clone RAM34), anti-Sca-1-PE-Cy7 (clone D7), anti-c-Kit-PE (clone 2B8) [27 (link)]. For CMP, MEP and GMP analyses, marrow cells were labeled with anti-FCγR-eFluor®450 (clone 93), anti-IL-7Rα-APC (clone A7R64), Lin-APC, anti-c-Kit-PE (clone 2B8), anti-CD34-FITC (clone RAM34) [28 (link)]. All antibodies were purchased from eBioscience (San Diego, Ca, USA). Samples were analyzed using a LSR II analyzer and FACSDIVA 6.1 software (BD Biosciences, San Jose, CA, USA); 3×10⁵ live cell events were recorded to analyze HSC, CMP, MEP and GMP frequency. For fibrillin-1 and macrophage visualization paraffin sections of tibiae from 3-month- old WT and Fbn1^Prx1−/− mice were incubated with anti-fibrillin-1 (a kind gift of Dr. L. Sakai) and anti F4/80 (clone BM8, Biolegend, San Diego, Ca, USA) whereas Alexa Fluor® 568 and 488 anti-rabbit antibodies (Molecular Probes, Eugene, OR, USA) were used for immunofluorescent labeling.

Smaldone S., Bigarella C.L., del Solar M., Ghaffari S, & Ramirez F. (2015). Fibrillin-1 microfibrils influence adult bone marrow hematopoiesis. Matrix biology : journal of the International Society for Matrix Biology, 52-54, 88-94.

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Isolation and Characterization of Stromal Vascular Fraction

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SVF was prepared as reported^{55 (link),59 (link),60 (link)}. Briefly, inguinal SAT or VAT were isolated from euthanized mice, minced and digested for 60 min in DMEM containing collagenase type I (2 mg/ml per gram of tissue; Invitrogen) at 37°C. Cell suspensions were then filtered through a 40-μm cell strainer and the SVF was collected as a pellet after centrifugation at 500g for 5 min. After RBC lysis, cells were stained with anti-CD45 (30-F11, BD), anti-F4/80 (clone BM8, BioLegend), anti-CD11b (M1/70, BD), anti-CD11c (HL3, BD), anti-CD206 (c06802, BioLegend; MR5D3, Acris), anti-TNF (MP6-XT22, BioLegend), anti-iNOS (CXNFT, eBioscience) and anti-CD49d [clone PS/2, AbD Serotec or clone 9C10(MFR4.B), BioLegend]. Cells were analyzed in a FACS Canto II flow cytometer (BD).

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Flow Cytometric Analysis of Immune Cells

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Single cell suspensions from blood, spleen, or tumor were prepared for flow cytometric analysis. Red blood cells in blood were lysed using ACK Lysis Buffer (Life Technologies). Cells were incubated with anti-Fc receptor antibody (clone 2.4G2, BD Biosciences) in Phosphate Buffered Saline (PBS) with 2% fetal bovine serum for 30 mins. Then, surface staining using the following monoclonal antibodies was performed: anti-CD8 (clone 53–6.7, BD Biosciences), anti-CD45 (clone 30-F11, Invitrogen), anti-CD90.1 (clone Ox-7, Biolegend), anti-CD62L (clone MEL-14, Biolegend), anti-CD25 (clone PC61, Biolegend), anti-Ly6C (clone HK1.4, Biolegend), anti-CD11b (clone M1/70, BD Biosciences), anti-CD11c (clone HL3, BD Biosciences), anti-I-A^b (clone AF6–120.1, Biolegend), anti-CD24 (clone M1/69, BD Biosciences), and anti-F4/80 (clone BM8, Biolegend). DAPI or LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Thermo Fisher)-stained cells were excluded from analysis. Samples were analyzed using LSR II (BD Biosciences) or LSRFortessa (BD Biosciences) with FlowJo software (TreeStar).

Oba T., Hoki T., Yamauchi T., Keler T., Marsh H.C., Cao X, & Ito F. (2020). A critical role of CD40 and CD70 signaling in cDC1s in expansion and antitumor efficacy of adoptively transferred tumor-specific T cells. Journal of immunology (Baltimore, Md. : 1950), 205(7), 1867-1877.

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Murine Peritoneal Cell Profiling

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Mice were euthanized with CO₂ as described above. Peritoneal exudate was taken from four CLP + Vehicle and five CLP + RvE1 mice. Peritoneal cells were differentiated using anti-CD11b (clone M1/70; eBioscience), anti-F4/80 (clone BM8; BioLegend), anti-Ly6G (clone 1A8; BioLegend), anti-I-A/I-E (clone M5/114.15.2; BioLegend), anti-Ly6C (clone HK1.4; BioLegend), anti-CD64 (clone X54-5/7.1; BioLegend) antibodies. Zombie NIR™ Fixable Viability Kit (Biolegend) was used to identify live cells. Cell counts were determined using Precision Count Beads (Biolegend). Ten thousand live cell events were acquired with a FACSCalibur (Becton Dickinson) and analyzed using FlowJo analysis software (version 9.2, Treestar Inc.).

Chen J., Purvis G.S., Collotta D., Al Zoubi S., Sugimoto M.A., Cacace A., Martin L., Colas R.A., Collino M., Dalli J, & Thiemermann C. (2020). RvE1 Attenuates Polymicrobial Sepsis-Induced Cardiac Dysfunction and Enhances Bacterial Clearance. Frontiers in Immunology, 11, 2080.

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Lung Immune Cell Profiling Post-Influenza

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BALF was collected on days 0, 7, and 8 post-influenza virus infection by flushing the lungs twice with 1 ml PBS. BALF was centrifuged, and the cell pellet was used for cell analysis. Cells were treated with Fc block (BD Biosciences) and surface stained with the following antibodies (at 1 µg/ml): anti-CD11b (clone M1/70, BioLegend), anti-CD11c (clone HL3; BD Biosciences), anti-F4/80 (clone BM8; BioLegend), and anti-Ly6G (clone 1A8; BioLegend). Data were acquired for equivalent numbers of cells on a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software version 9. Cell types were defined as previously described (24 (link)). Briefly, neutrophils were defined as CD11b^hi Ly6G^hi cells, and alveolar macrophages were defined as CD11c^hi F4/80^hi CD11b⁻ cells.

Karlsson E.A., Meliopoulos V.A., van de Velde N.C., van de Velde L.A., Mann B., Gao G., Rosch J., Tuomanen E., McCullers J., Vogel P, & Schultz-Cherry S. (2017). A Perfect Storm: Increased Colonization and Failure of Vaccination Leads to Severe Secondary Bacterial Infection in Influenza Virus-Infected Obese Mice. mBio, 8(5), e00889-17.

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Tissue Histology Analysis by H&E, Iron, and IHC

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For Hematoxylin and Eosin (H&E) staining, tissues were fixed for 24 h in 10% neutral buffered formalin, dehydrated and embedded in paraffin. Tissue sections (3–5 µm) were stained with H&E. Images were acquired with KFBIO KF-PRO-120 digital pathology slide scanner.
The frozen tissues were sliced into sections of 10 μm each at −20 °C and fixed for 10 min in 4% paraformaldehyde. Tissue sections were staining with Perls’ Iron Stain Kit (Solarbio, Beijing, China) following manufacturer’s instructions. When indicated, Perls’ staining was further enhanced using the DAB-kit (Beyotime, Shanghai, China). For immunohistochemistry, tissue sections were fixed, washed in PBS and treated with H₂O₂ to block endogenous peroxidase. Sections were incubated with anti-F4/80 (clone BM8, Biolegend, San Diego, CA, USA, 1:500) at 4 ℃ overnight and were incubated with HRP-labeled Goat Anti-Rat IgG (H + L) (Beyotime, Shanghai, China, 1:100) at 37 °C for 1 h and then stained with diaminobenzidine (DAB) at room temperature for 10 min in the dark, followed by counterstaining with hematoxylin for cell nuclei. Images were acquired with a Leica microscope.

Wang S., Cheng M., Peng P., Lou Y., Zhang A, & Liu P. (2021). Iron Released after Cryo-Thermal Therapy Induced M1 Macrophage Polarization, Promoting the Differentiation of CD4+ T Cells into CTLs. International Journal of Molecular Sciences, 22(13), 7010.

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Multiparameter Flow Cytometry of Immune Cells

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Single cell suspensions (1 × 10⁶ cells/100 μl) were prepared with dead cells excluded by using Zombie Aqua Fixable Viability Kit (Biolegend, USA). After being washed, cells were resuspended in buffer (PBS containing 5% FBS) and were blocked in Fc block (Anti-mouse CD16/32, Clone 2.4G2, BD, USA) at 4°C for 30 min. Cells were then incubated with following various antibodies for 30 min at 4°C. anti-CD11b (clone M1/70, Biolegend, USA), anti-F4/80 (clone BM8, Biolegend, USA), anti-Ly-6G/Ly-6C (clone RB6-8C5, eBioscience, USA), anti-CD11c (clone N418, Biolegend, USA), anti-I-A/I-E (clone M5/114.15.2, Biolegend, USA); Rat IgG2b,ҝ Isotype Ctrl (Biolegend, USA); anti-CD3 (clone 17A2, Biolegend, USA), Anti-CD4 (clone RM4-5, BD, USA), anti-CD8α (clone 53–6.7, BD, USA), anti-CD44 (clone IM7, BD, USA); anti-CD62L (clone MEL-14, eBioscience, USA), anti-Ki67 (clone SolA15, eBioscience, USA). Ki67 staining was implemented using Foxp3/Transcription Factor Staining Buffer Set (eBioscience, USA) according to manufacturers’ instructions.

Wei S., Huang J., Liu Z., Wang M., Zhang B., Lian Z., Guo Y, & Han H. (2019). Differential immune responses of C57BL/6 mice to infection by Salmonella enterica serovar Typhimurium strain SL1344, CVCC541 and CMCC50115. Virulence, 10(1), 248-259.

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