F4 80 apc bm8

Manufactured by BioLegend

F4/80-APC (BM8) is a monoclonal antibody that binds to the F4/80 antigen, a 160 kDa glycoprotein expressed on the surface of mature mouse macrophages. The antibody is conjugated to the fluorescent dye allophycocyanin (APC), which allows for the detection and analysis of F4/80-positive cells by flow cytometry.

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F4/80 (349 citations) F4/80-APC (73 citations) F4/80 (BM8, (56 citations) APC anti-mouse F4/80 Antibody (BM8, (4 citations) APC anti-mouse F4/80 (BM8, (4 citations) F4/80 APC (clone BM8, (3 citations) Anti-F4/80 APC-Cy7 (clone BM8, (3 citations) APC anti-mouse F4/80 Clone BM8 (3 citations) F4/80 (APC/Cy7; clone BM8) (2 citations) APC-Cy7-anti-mouse-F4/80 (BM8, (2 citations)

8 protocols using f4 80 apc bm8

Multiparametric Flow Cytometry of Influenza Vaccine

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The H5N1 HA (259–274) (SNGNFIAPEYAYKIVK) and OVA (326–339) (AVHAAHAEINEAGR) were synthesized by [Peptide 2.0] with >95% purity. The inactivated influenza vaccine, A/H5N1 Influenza Vaccine, was obtained from [beiresources.org]. The HA protein was obtained from [eEnzyme]. Percp-Cy55-CD19(6D5), Brilliant Violet 421-CD44(IM7), Brilliant Violet 421-CXCR5(L138D7), Alexa Fluor 700-CD4(GK1.5), Alexa Fluor 700-IgD(11-26c.2a), FITC-CD80(16-10A1), PE-PD-L2(TY25),PE-GL7(GL7), PE-Cy7-IgM(RMM-1), PE-Cy7-CD86(GL-1), PE-Cy7-CD69(H1.2F3), PE-Cy7-PD-1(29F.1A12), PE-Cy7-CD28(37.51), APC-CD95(SA367H8), APC-CD40(3/23), APC-B220(RA3-6B2), APC-CD11c(N418) and APC-F4/80(BM8) were from [Biolegend]. Brilliant Ultra Violet 395-CD3 (17A2) and Brilliant Ultra Violet 496-CD4 (RM4-4) were from [BD Biosciences]. Fixable viability dye eFluor 780 was from [eBioscience]. Celltrace CFSE proliferation dye and Celltrace violet dye were from [Thermo Fisher Scientific]. PE-CD99 (polyclonal) was from [R&D systems].

Song N., Welsh R.A, & Sadegh-Nasseri S. (2023). Proper development of long-lived memory CD4 T cells requires HLA-DO function. Frontiers in Immunology, 14, 1277609.

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Multiparameter Flow Cytometry and Immunofluorescence

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The following Ab were used for flow cytometry: Phycoerythrin (PE)-conjugated anti-CD25, fluorescein isothiocyanate (FITC)-conjugated anti-CD69, PE-conjugated anti-mouse CD3e (clone eBio500A2), APC-conjugated anti-mouse IFN-γ (clone XMG1.2) were all purchased from eBioscience. Alexa Fluor® 488-anti-mouse CD4 (RM4-5), PE-anti-mouse CD3e (145-2C11), PerCP/Cy5.5-anti-mouse CD8a (53-6.7), APC-anti-mouse CD45 (30-F11), and APC-F4/80 (BM8) were obtained from Biolegend (San Diego, CA). FITC-anti-mouse CD11b (M1/70) was purchased from BD Bioscience (Mississauga, ON).
The following Ab were used for immunofluorescence: an unconjugated primary mouse anti-CSPG polyclonal Ab was obtained from Sigma-Aldrich Canada. An unconjugated rabbit anti-Iba-1 polyclonal Ab was obtained from Wako Chemicals (Wako, TX). Conjugated fluorescent secondary donkey anti-mouse IgG H&L (Alexa Fluor® 555) and donkey anti-rabbit IgG H&L (Alexa Fluor® 488, pre-adsorbed) Abs were obtained from Abcam (Cambridge, UK).

Warford J.R., Lamport A.C., Clements D.R., Malone A., Kennedy B.E., Kim Y., Gujar S.A., Hoskin D.W, & Easton A.S. (2018). Surfen, a proteoglycan binding agent, reduces inflammation but inhibits remyelination in murine models of Multiple Sclerosis. Acta Neuropathologica Communications, 6, 4.

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Flow Cytometry Analysis of Bone Marrow and Spleen Cells

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Bone marrow and spleen cells were stained for flow cytometry as previously described (Mayer et al., 2017 (link)). Gating strategy for BM erythroid cells was described in the Supplementary Figure 1. Cells were stained with the following antibodies: CD45-FITC (30-F11, eBioscience), CD11b-FITC (M1/70, eBioscience), Gr-1-FITC (RB6-8C5, eBioscience), Ter119-APC (TER119, Biolegend) and CD44-PE (IM7, Biolegend), F4/80-APC (BM8, Biolegend), Gr-1-FITC (RB6-8C5, Biolegend), CD3-PE (145-2C11, Biolegend), Ly6C-PE (HK1.4, Biolegend), CD106-PE (429, Biolegend), CD45R/B220-APC (RA3.682, Biolegend). Cells were analyzed using AccuriC6 flow cytometer and data were analyzed using FlowJo software (v10.7.1).

Myneni V.D., Szalayova I, & Mezey E. (2021). Differences in Steady-State Erythropoiesis in Different Mouse Bones and Postnatal Spleen. Frontiers in Cell and Developmental Biology, 9, 646646.

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Immunofluorescence Staining of Glioma Samples

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Brains from perfused A2.DR1 glioma-bearing mice were stored in Tissue-Tek OCT compound (Sakura) at −80 °C and cut into 6- to 8-μm slices using a cryotome (Leika). For immunofluorescence staining, slides were air dried, fixed with ice-cold methanol, and blocked for 2 hours with normal goat serum (Sigma). Slides were incubated overnight with the respective primary antibody (polyclonal Cic AB, 1:1000, Abcam; DRB1.01, L243, BioLegend) in blocking buffer. The slides were then incubated with the respective primary or secondary antibody for 1 hour (F4/80-APC, BM8, BioLegend, 1:200; goat-anti-rabbit, 1:300, AF488, Thermo Fisher Scientific; goat-anti-mouse AF488, 1:300, Thermo Fisher Scientific) and mounted using DAPI-containing mounting medium (Invitrogen). Images were acquired within 6 hours on a Cellobserver (Zeiss) or LSM700 confocal microscope (Zeiss).
Hematoxylin and eosin (H&E) staining was performed as previously described (6 (link)). Briefly, 8-μm slides were fixed with Roti-Histofix 4.5%. H&E staining was performed using hematoxylin and bluing reagent for 4 minutes.

Kilian M., Friedrich M., Sanghvi K., Green E., Pusch S., Kawauchi D., Löwer M., Sonner J.K., Krämer C., Zaman J., Jung S., Breckwoldt M.O., Willimsky G., Eichmüller S.B., von Deimling A., Wick W., Sahm F., Platten M, & Bunse L. (2021). T-cell Receptor Therapy Targeting Mutant Capicua Transcriptional Repressor in Experimental Gliomas. Clinical Cancer Research, 28(2), 378-389.

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Isolation and Characterization of Immune Cells

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Spleens were minced through a 70 μm sieve and leukocytes were obtained after lysing red blood cells with ACK buffer. Livers were minced through a 100 μm cell strainer and digested for 30 min at 37 °C with rotation in HBSS (Ca²⁺, Mg²⁺) buffer containing 1 mg/ml (335 U/mg) collagenase IV (Sangon Biotech, Shanghai, China) and 200 μg/ml (100 Kunitz U/ml) DNase I (Sangon Biotech, Shanghai, China). Liver leukocytes were separated by percoll gradient centrifugation (GE Healthcare, Freiburg, Germany). The cells were then resuspended in RPMI-1640 at 1 × 10⁶/ml, and surface staining was performed as previously described [16 (link)].
F4/80 and CD11b were used to stain macrophages, CD11c and CD11b for DCs, and Ly6G and CD11b for granulocytes. CD11c-FITC (N418; Biolegend), Gr1-PE (RB6-8C5; Biolegend), CD11b-PerCP-Cy5.5 (M1/70; eBioscience), F4/80-APC (BM8; Biolegend), and CD3-Pacific Blue (145-2C11; eBioscience) antibodies were utilized. As for intracellular iNOS-PE (W16030C; Biolegend) staining, the fixation and permeabilization steps were followed by Intracellular Fixation & Permeabilization Kit (eBioscience). Flow cytometric acquisition was performed with a BD FACSVerse flow cytometer, and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

OuYang X., Liu P., Zheng Y., Jiang H., Lv Q., Huang W., Hao H., Pian Y., Kong D, & Jiang Y. (2023). TRIM32 reduced the recruitment of innate immune cells and the killing capacity of Listeria monocytogenes by inhibiting secretion of chemokines. Gut Pathogens, 15, 32.

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Immune Cell Profiling in Cellular Signaling

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Escherichia coli O55:B5 LPS and oleic acid were purchased from Sigma-Aldrich. Everolimus and NVP-BEZ235 were provided by Novartis. Phospho-S6 (Ser240/244) and phospho-Akt (Ser473) were purchased from Cell Signaling Technology. TER-119-APC, CD4-APC (RM4-5), CD8a-PerCP (53-6.7), CD19-FITC (6D5), I-Ab-PE (AF6-120.1), CD11c-FITC (N418), and F4/80-APC (BM8) antibodies for flow cytometry were purchased from BioLegend.

Üstün S., Lassnig C., Preitschopf A., Mikula M., Müller M., Hengstschläger M, & Weichhart T. (2015). Effects of the mTOR inhibitor everolimus and the PI3K/mTOR inhibitor NVP-BEZ235 in murine acute lung injury models. Transplant immunology, 33(1), 45-50.

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Evaluating pHLIP Peptide Specificity

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To determine the specificity of pHLIP delivery, animals were injected intravenously with pHLIP-A750-Var3 or the non-inserting control peptide pHLIP-A750–5K-Var3 (4 nmol). Animals were sacrificed after 4, 12 and 24 hours, and uptake in different tissues was determined by near-infrared fluorescence imaging, performed on an IVIS Spectrum system (Caliper Life Science) with appropriate excitation (Ex) and emission (Em) filter sets (Ex/Em = 745/800 nm). For flow cytometry analysis, entire aortas (from the root to the iliac bifurcation) were harvested 4 hours after injection with 4 nmol A546-Var3–5K or A546-Var3. The aortas were cut into small pieces and subjected to enzymatic digestion with 400 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNAse I and 60 U/ml hyaluronidase (Sigma-Aldrich) for 1 h at 37ºC while shacking. Macrophages and monocytes were identified with the following antibodies (all from Biolegend): Lineage-PE (CD3, CD90.2 clone 53–2.1, CD19, Ly6G clone 1A8, NK1.1 clone PK126, Ter119 clone Ter119, CD11c clone HL3), CD11b-PacificBlue clone M1/70, F4/80-APC (BM8) and Ly6-C-FITC (clone AL-21). Flow cytometry was performed using a BD LSRII (BD Biosciences), and data were analyzed using FlowJo software v8.7 (Tree Star, Inc.).

Zhang X., Rotllan N., Canfrán-Duque A., Sun J., Toczek J., Moshnikova A., Malik S., Price N.L., Araldi E., Zhong W., Sadeghi M.M., Andreev O.A., Bahal R., Reshetnyak Y.K., Suárez Y, & Fernández-Hernando C. (2022). Targeted Suppression of miRNA-33 Using pHLIP Improves Atherosclerosis Regression. Circulation research, 131(1), 77-90.

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Quantifying Bacterial Loads and Immune Cells

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Bacterial loads were determined in BALF and blood. Serial dilutions of samples were plated on blood agar and colony forming units (CFU) were counted. To test for cell recruitment, cells were enriched by spinning the BALF (400 g 10 min RT), incubated with blocking antibody (anti-CD16/32, Biolegend) and stained using CD11c-FITC (N418, Biolegend), SiglecF-PE (E50-2440, BD bioscience), Ly6C-PerCp (HK1.4, Biolegend), F4/80-APC (BM8, Biolegend), CD45-Alexa700 (30-F11, Biolegend), Ly6G-BV421 (1A8, Biolegend) and CD11b-BV510 (M1/70, Biolegend).

Kostadinova E., Chaput C., Gutbier B., Lippmann J., Sander L.E., Mitchell T.J., Suttorp N., Witzenrath M, & Opitz B. (2016). NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence. Scientific Reports, 6, 30943.

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