Anti cd11b bv421

Manufactured by BioLegend

Sourced in United States, Germany

Anti-CD11b-BV421 is a fluorochrome-conjugated monoclonal antibody that binds to the CD11b antigen. CD11b is a cell surface glycoprotein expressed on the surface of various immune cells, including monocytes, macrophages, and neutrophils. The BV421 fluorochrome is used to label the antibody.

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CD11b (457 citations) Anti-CD11b (179 citations) CD11b-BV421 (26 citations) BV421-conjugated anti-CD11b (4 citations) Anti‐CD11b BV421 (clone M1/70 (2 citations) Anti-mouse CD11b-BV421 (2 citations)

9 protocols using anti cd11b bv421

Isolation and Characterization of Mouse Immune Cells

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The peripheral blood was obtained through the jugular vein of mice and mononuclear cells
were isolated through erythrocyte lysis. The cells were stained with anti-mCD45-FITC,
anti-CD45.2-BV711 (BD Bioscience, East Rutherford, NJ, USA), anti-CD3-PE, anti-CD3-APC,
anti-CD19-PE/Cy7, anti-CD11b-BV421 and anti-Gr-1-BV510 (BioLegend, San Diego, CA, USA) to
identify lymphoid and myeloid lineages of cells. Anti-CD132-PE (clone TUGm2, BioLegend)
was used to detect IL2RG protein. Flow cytometry was performed with BD LSR Fortessa II,
The BD FACS Aria Fusion (BD Bioscience).

Byambaa S., Uosaki H., Hara H., Nagao Y., Abe T., Shibata H., Nureki O., Ohmori T, & Hanazono Y. (2019). Generation of novel Il2rg-knockout mice with clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9. Experimental Animals, 69(2), 189-198.

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Characterizing Intestinal Immune Cells

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Small intestinal lamina propria cells were resuspended in a 1:50 dilution of F_C block [BD Pharmingen; Clone 2.4G2] in FACS buffer containing 5 mM EDTA and incubated on ice for 10 min. Cells were then stained with fluorophore-conjugated primary antibodies for 30 min on ice before fixing with 2% PFA: anti-CD45 FITC [BioLegend; clone 30-F11], anti-CD19 APC [BD; clone 1D3], anti-CD3 Alexa Fluor 700 [BioLegend; clone 17A2], anti-Ly6G BV510 [BioLegend; clone 1A8], anti-F4/80 PE [BioLegend; clone BM8] and anti-CD11b BV421 [BioLegend; clone M1/70]. Splenocytes were used for single colour controls. Samples were run on a NovoCyte Advanteon Flow Cytometer system [Agilent] and data analysis was performed using FlowJo [version 10.7.1].

Chakraborty R., Maltz M.R., Del Castillo D., Tandel P.N., Messih N., Anguiano M, & Lo D.D. (2021). Selective Targeting of Tumour Necrosis Factor Receptor 1 Induces Stable Protection from Crohn’s-Like Ileitis in TNFΔARE Mice. Journal of Crohn's & Colitis, 16(6), 978-991.

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Murine Macrophage Activation and Infection

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Macrophages were derived from the bone marrow of naïve mice in IMEM medium (Life Technologies) supplemented with 10% FBS, pen/strept, L-glutamine, and 15% L929 cell-conditioned medium as a source of colony-stimulating factor-1 (CSF-1). Cells were then left for 6 days at 37°C in a 5% CO₂ incubator. For one set of experiments, BMM were washed and resuspended in supplemented DMEM. Cells were classically activated (M1) with 20 ng/ml of murine IFNγ (Peprotec), alternatively activated (M2) with 20 ng/ml of murine IL-4 (Peprotec), or infected with L. donovani amastigotes at a MOI of 1:10. BMM were then incubated for 24h and stained with anti-CD11b-BV421, iNOS-APC (Biolegend), and CD38-BV711 (eBioscience) or processed for qPCR analysis. Samples were acquired on a BD LSRFortessa cell analyzer (Becton Dickinson).

Hammami A., Abidin B.M., Charpentier T., Fabié A., Duguay A.P., Heinonen K.M, & Stäger S. (2017). HIF-1α is a key regulator in potentiating suppressor activity and limiting the microbicidal capacity of MDSC-like cells during visceral leishmaniasis. PLoS Pathogens, 13(9), e1006616.

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Multicolor Flow Cytometry Immune Profiling

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All fluorophore-conjugated antibodies were obtained from Biolegend: anti-CD45-FITC, anti-CD206-PE, anti-F4/80-PE/Cy7, anti-CD115-APC, anit-IA/IE-APC/Cy7, anti-CD3-BV421, anti-FoxP3-FITC, anti-CD45-PE, anti-CD4-PE/Cy7, anti-RoRγt-PE/Cy7, anti-CD8a-APC/Cy7, anti-Granzyme B-FITC, anti-PD-1-APC, and anti-CD11b-BV421. Cell staining was performed for 1 hour at 4 °C, with 2% rat serum (Sigma) to reduce antibody binding to Fc receptors. In all conditions, doublets and multiplets were excluded by forward scatter pulse width (FSC-W) vs. forward scatter pulse area (FSC-A) gating and live cells were selected via negative selection for Yellow Amine Dye (Life Technologies) staining. Gating of positively stained cells was determined by fluorescence-minus-one (FMO) controls. Cells were analyzed using an 8-color MACSQuant 10 (Miltenyi Biotec) with three laser sources (405 nm, 488 nm, 635 nm) and FlowLogic 501.2 A software (Inivai Technologies).

Ball M.S., Bhandari R., Torres G.M., Martyanov V., ElTanbouly M.A., Archambault K., Whitfield M.L., Liby K.T, & Pioli P.A. (2020). CDDO-Me Alters the Tumor Microenvironment in Estrogen Receptor Negative Breast Cancer. Scientific Reports, 10, 6560.

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Multicolor Flow Cytometry and Molecular Analyses of Immune Cells

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For flow cytometric analysis, anti-CD45.2-V500(562129, BD bioscience, 1:100), anti-CD45.2-APC(109814, BioLegend, 1:100), anti-CD4-PerCP/Cy5.5(100433, BioLegend, 1:100), anti-CD8a-PerCP/Cy5.5(100733, BioLegend, 1:100), anti-CD11b-PerCP/Cy5.5(45-0112-80, eBioscience, 1:100), anti-Ly6G-PerCP/Cy5.5(127615, BioLegend, 1:100), anti-B220-PerCP/Cy5.5(103235, BioLegend, 1:100), anti-CD11b-BV421(101251, BioLegend, 1:100), anti-CD64-APC(139306, BioLegend, 1:100), anti-F4/80-PE(123110, BioLegend, 1:100), anti-Ly6c-PE-Cy7(128018, BioLegend, 1:100) were used. For western blotting, anti-Cx40(AB1726, Merck Millipore, 1:500), anti-Cx43(C6219, Sigma, 1:2000), anti-Cx45(AB1745, Merck Millipore, 1:2000), anti- α -tubulin(T6199, Sigma, 1:1000), anti-rabbit IgG-HRP(7074, Cell Signaling Technology, 1:5000), anti-mouse IgG-HRP(7076, Cell Signaling Technology, 1:5000) were used. For immunohistochemistry, anti-F4/80(MCA497G, Serotec, 1:500), anti-Ly6G(127601, BioLegend, 1:500), anti-B220 (103201, BioLegend 1:500), anti-CD3(GTX42110, GeneTex, 1:500), anti-Cx43(C6219, Sigma, 1:2000), anti-N-cadherin(33-3900, ThermoFisher, 1:500) were used. The second antibodies used were Alexa-Fluor-488-conjugated anti-mouse-IgG(A-11001, ThermoFisher, 1:2000) and Alexa-Fluor-635-conjugated anti-rabbit-IgG(A-31576, ThermoFisher, 1:2000).

Sugita J., Fujiu K., Nakayama Y., Matsubara T., Matsuda J., Oshima T., Liu Y., Maru Y., Hasumi E., Kojima T., Seno H., Asano K., Ishijima A., Tomii N., Yamazaki M., Kudo F., Sakuma I., Nagai R., Manabe I, & Komuro I. (2021). Cardiac macrophages prevent sudden death during heart stress. Nature Communications, 12, 1910.

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Multicolor Flow Cytometry Analysis of Immune Cells and Platelet Activation

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Peripheral blood was obtained through a jugular vein and mononuclear cells were isolated through erythrocyte lysis. The cells were stained with anti-mouse CD45-fluorescein isothiocyanate (FITC) (BD PharMingen), anti-CD3-PE, anti-CD19-PE/Cy7, anti-CD11b-BV421, anti-Gr-1-BV510, and anti-CD132-APC (BioLegend) to identify lymphoid and myeloid lineages. Washed and activated platelets were stained with Brilliant Violet 421-labeled anti-mouse CD41 (BioLegend), DyLight 649-labeled rat anti-mouse GPIbα (CD42b), phycoerythrin (PE)-labeled anti-activated integrin αIIbβ3 (JON/A), and FITC-labeled anti-mouse CD62P (Anti-P-Selectin) (Emfret Analytics, Eibelstadt, Germany) antibodies. Flow cytometry was performed with BD LSRFortessa II.

Byambaa S., Uosaki H., Ohmori T., Hara H., Endo H., Nureki O, & Hanazono Y. (2021). Non-viral ex vivo genome-editing in mouse bona fide hematopoietic stem cells with CRISPR/Cas9. Molecular Therapy. Methods & Clinical Development, 20, 451-462.

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Immunoblotting, Immunofluorescence, and Flow Cytometry Analysis

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The following antibodies were used for immunoblotting, immunofluorescence, and flow cytometry analysis: anti-Caspase-1 (ab179515; Abcam), anti-NLRP3 (ab263899; Abcam), anti-ASC (sc514414; Santa Cruz), anti-NF-κB P50 (ab32360; Abcam), anti-NF-κB P65 (cst6956; Cell Signaling Technology), anti-phospho–NF-κB P65 (cst3033; Cell Signaling Technology), anti-MyD88 (ab219413; Abcam), anti-IKK-β (ab124951; Abcam), anti-TRAF6 (ab33915; Abcam), anti-GPR81 (ab106942; Abcam), anti-tubulin (11224; Proteintech), anti-pro-IL-1β (cst12242; Cell Signaling Technology), anti–F4/80-PE (123110; BioLegend), anti–CD11b-BV421 (101236; BioLegend), anti–CD68-APC (137007; BioLegend), anti–CD206-AF647 (141712; BioLegend), anti–Ly6G-FITC (11–9668-80; eBioscience), anti-CD16/32 (14–0161-82; eBioscience), anti-CD11b (2185-1-ap; Proteintech), anti-F4/80 (cst70076; Cell Signaling Technology), anti-Ly6G monoclonal antibody (65140-1-ig; Proteintech).
The following reagents were used for this study: caerulein (C9026-1 MG; Sigma, Munich, Germany), ELISA kit for IL-1β, IL-6 and TNF-α (Lianke), alpha-amylase determination kit (BIOSINO, Beijing, China), PBMC isolation kit (LTS1092; TBD), LPS from Escherichia coli O55:B5 (Sigma, Germany), L-lactate assay kit (ab65330; Abcam), sodium L-lactate (L7022; Sigma), L-lactic acid (L1750; Sigma).

Li H., Xie J., Guo X., Yang G., Cai B., Liu J., Yue M., Tang Y., Wang G., Chen S., Guo J., Qi X., Wang D., Zheng H., Liu W., Yu H., Wang C., Zhu S.J, & Guo F. (2022). Bifidobacterium spp. and their metabolite lactate protect against acute pancreatitis via inhibition of pancreatic and systemic inflammatory responses. Gut Microbes, 14(1), 2127456.

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Microglial and Macrophage Cytokine Profiling

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Direct ICS was performed as previously described (Cusick et al., 2013 (link); Liu and Whitton, 2005 ). Briefly, animals were retro-orbitally injected with 250 μg Brefeldin A (SigmaAldrich), an inhibitor of protein transport, 6 hours before being sacrificed. Brains were immediately collected and processed on ice. Extracellular staining was carried out using anti-CD45-V500 (BD Biosciences, San Jose, CA) and anti-CD11b-BV421 (BioLegend, San Diego, CA) in order to distinguish microglial (CD45^low/intCD11b⁺), macrophage (CD45^hiCD11b⁺), and lymphocyte (CD45^hiCD11b⁻) cell types. Cells were then treated with cytoperm/cytofix (BD Biosciences) and stained with anti-TNF-α-Pe-Cy7 (BioLegend) and anti-IL-6-PE (eBioscience). Gating for all flow cytometry experiments was determined by Fluorescence-minus-one (FMO) for CD45, CD11b, TNF-α, and IL-6 antibodies.

Hanak T.J., Libbey J.E., Doty D.J., Sim J.T., DePaula-Silva A.B, & Fujinami R.S. (2018). Positive Modulation of mGluR5 Attenuates Seizures and Reduces TNF-α+ Macrophages and Microglia in the Brain in a Murine Model of Virus-Induced Temporal Lobe Epilepsy. Experimental neurology, 311, 194-204.

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Annexin V-FITC Apoptosis, Cell Cycle, and Differentiation Analysis

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The Annexin V-FITC Apoptosis Detection kit (Dojindo) was used for apoptosis assays. Cells were harvested and washed twice with cold PBS. After being suspended in 1 × Annexin V binding buffer, cells were stained with Annexin V-FITC and propidium iodide (PI) for 15 min on ice. For cell cycle assays, a DNA labeling solution (Cytognos) was used. Briefly, after collection and washing with cold PBS, cells were stained with DNA labeling solution for 15 min at 25 °C. For cell differentiation assays, human AML cells were harvested and washed with cold PBS; peripheral blood (PB), BM, and spleen cells were harvested from AML1-ETO9a AML mice, and erythrocytes were lysed using RBC Lysis Buffer (00-4333-57, Invitrogen). Human AML cells were stained with anti-human CD11b (301310, Biolegend) and isolated mice cells were stained with anti-CD11b-BV421 (101251, Biolegend) for 30 min on ice. Stained cells were analyzed using a CytoFLEX S Flow Cytometer (Beckman Coulter).

Zhou W., Li S., Wang H., Zhou J., Li S., Chen G., Guan W., Fu X., Nervi C., Yu L, & Li Y. (2024). A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia. Experimental Hematology & Oncology, 13, 9.

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