Clone m1 70

Manufactured by BioLegend

Sourced in United States

The Clone M1/70 is a monoclonal antibody used for research purposes. It is designed to recognize and bind to the CD11b antigen, which is expressed on the surface of myeloid cells, including monocytes, macrophages, and granulocytes. The Clone M1/70 antibody can be used in various applications, such as flow cytometry and immunohistochemistry, to identify and study these cell populations.

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26 protocols using clone m1 70

Tumor-Infiltrating Lymphocyte Analysis

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The significant differences in tumor volume between IDO-APT treated group and Scr-APT treated group began to appear Mice were euthanized 2 days after the third treatment and the tumor tissue was collected. Tumor tissues were cut into small pieces and digested with collagenase D and DNase I for 30 min at 37°C. After grinding and passing through the 200 meshes strainer, TILs were isolated from the dissociated cells by Ficoll-Hypaque (Dakewe Biotech). For intracellular cytokine analysis, cells were re-stimulated with 100 ng/mL PMA and 500 ng/mL ionomycin in the presence of protein transport inhibitor cocktail for 5 h. After staining with anti-CD45-PE-Cy7 and anti-CD8-FITC, cells were fixed and permeabilized and then stained with anti-TNF-α-PE or anti-IFN-γ-APC. For T_reg cells analysis, cells were stained with anti-CD45-PE-Cy7, anti-CD4-FITC, anti-CD25-PE, and anti-Foxp3-APC. For tumor-associated macrophages analysis, cells were stained with anti-CD45-PEcy7, anti-CD11b-FITC (Cat 101205, Clone M1/70, Biolegend), and anti-F4/80-APC (Cat 123115, Clone BM8, Biolegend). For granulocytic or monocytic MDSC analysis, cells were stained with anti-CD45-PEcy7, anti-CD11b-FITC, anti-Ly6C-PE (Cat 128007, Clone HK1.4, Biolegend) or anti-Ly6G-PerCP (Cat 127653, Clone 1A8, Biolegend). The stained cells were then measured by flow cytometry.

Zhu Z., Yang Z., Zhu C., Hu Z., Jiang Z., Gong J., Yuan Y., Chen X., Jin Y, & Yin Y. (2023). Development of a DNA aptamer targeting IDO1 with anti-tumor effects. iScience, 26(8), 107367.

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Microglial Amyloid Uptake in 5xFAD Mice

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5xFAD mice (young adults: 10–12 weeks old; adults: 10–12 months old) were injected intraperitoneally with methoxy-XO4 (10 mg kg⁻¹ body weight, Tocris, cat. no. 4920). After 3 h, mice were transcardially perfused with ice-cold 1× PBS. Hippocampi were collected, and microglia were isolated by using density gradient separation and prepared as described previously with slight modifications^{10 (link)}. In addition to the microglia surface markers CD11b (1:200, clone M1/70, BioLegend, cat. no. 101212) and CD45 (1:200, clone 30-F11, BioLegend, cat. no. 103106), the following lineage markers were added: anti-CD3 (1:300, clone 17A2, BioLegend, cat. no. 100220), anti-CD19 (1:300, clone 6D5, BioLegend, cat. no. 115520), anti-CD45R (1:300, clone RA3-6B2, BD Biosciences, cat. no. 552772), Ly6C (1:300, clone AL-21, BD Biosciences, cat. no. 560593) and Ly6G (1:300, clone 1A8, BD Biosciences, cat. no. 560601) for 20 min at 4 °C. Percentage and MFI of methoxy-XO4-positive CD11b⁺CD45^low microglia were determined by flow cytometry using a FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star).

d’Errico P., Ziegler-Waldkirch S., Aires V., Hoffmann P., Mezö C., Erny D., Monasor L.S., Liebscher S., Ravi V.M., Joseph K., Schnell O., Kierdorf K., Staszewski O., Tahirovic S., Prinz M, & Meyer-Luehmann M. (2021). Microglia contribute to the propagation of Aβ into unaffected brain tissue. Nature Neuroscience, 25(1), 20-25.

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High-Throughput Screening for HoxA9 Inhibitors

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The primary screen was performed at the University of New Mexico Center for Molecular Discovery. Details of the primary and counterscreens have been described (Sykes et al., 2015 ). In brief, Lys-GFP-ER-HoxA9 cells (2,500) were seeded in a volume of 50 μl in each well of a tissue-culture-treated 384-well plate. Compounds were added to the well by robotic pinning to a final concentration of 4 μM. Given that there is no known small-molecule inhibitor of HoxA9, we took advantage of the ER-HoxA9 fusion protein and used the estrogen antagonist fulvestrant as a positive control at a concentration of 10 μM. Following a 4-day incubation, 10 μl of staining solution (anti-mouse-CD11b-APC antibody at a concentration of 1 to 100; Clone M1/70, BioLegend) and inert marker beads (Spherotech) were added to each well. Cells were analyzed by high-throughput flow cytometry (HyperCyt system, IntelliCyt). Live cells were distinguished from dead cells on the basis of forward and side scatter properties. Differentiation was assayed by measuring GFP (FL-1) and APC (FL-4) fluorescence.

Sykes D.B., Kfoury Y.S., Mercier F.E., Wawer M.J., Law J.M., Haynes M.K., Lewis T.A., Schajnovitz A., Jain E., Lee D., Meyer H., Pierce K.A., Tolliday N.J., Waller A., Ferrara S.J., Eheim A.L., Stoeckigt D., Maxcy K.L., Cobert J.M., Bachand J., Szekely B.A., Mukherjee S., Sklar L.A., Kotz J.D., Clish C.B., Sadreyev R.I., Clemons P.A., Janzer A., Schreiber S.L, & Scadden D.T. (2016). Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia. Cell, 167(1), 171-186.e15.

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Neutrophil Receptor Profiling by Flow Cytometry

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Bone marrow cells were flushed with ice-cold HBSS^–++, filtered through 40 μm cell strainers, counted, pelleted at 326 × g for 10 min at 4°C, and resuspended in ice-cold DPBS⁺⁺⁺⁺ at 4 × 10⁷ cells/ml. 125 μl of cells were either kept on ice, or were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF, or with 1 μg/ml LPS, for 45 min at 37°C. Cells were sedimented at 10,000 × g for 30 s at 4°C, resuspended in Fc block (BD Biosciences, clone 2.4G2, 1:1000; for Mac1 and L-selectin) or in DPBS⁺⁺⁺⁺ (for FcγRIII), and incubated on ice for 15 min. Cells were sedimented at 10,000 × g for 30 s, resuspended in ice-cold DPBS⁺⁺⁺⁺ containing fixable viability dye (eBioscience, eFluor™ 780, 1:1000), antibodies for neutrophil markers Ly6G (Ly6G-BV510, BioLegend, clone 1A8, 1:500) and Mac1 (CD11b-AF647, BD Bioscience Clone M1/70, 1:1000), and PE-labelled antibodies for FcγRIII (CD16, BioLegend, clone S17014E, 1:100) or L-selectin (BD Biosciences clone MEL-14, 1:100), and were incubated on ice for 30 min. Cells were washed in ice-cold HBSS^–++, 1 mM EDTA, resuspended in 300 μl ice-cold HBSS^–++, 1 mM EDTA, and kept on ice. Flow cytometry was performed using a BioRad ZE5 flow cytometer, recording 20,000 neutrophils per sample. Neutrophils were identified by Ly6G^hi, CD11b^hi staining, and the mean fluorescence intensity (mfi) of receptor levels on the neutrophil surface was quantitated using FlowJo.

Hornigold K., Chu J.Y., Chetwynd S.A., Machin P.A., Crossland L., Pantarelli C., Anderson K.E., Hawkins P.T., Segonds-Pichon A., Oxley D, & Welch H.C. (2022). Age-related decline in the resistance of mice to bacterial infection and in LPS/TLR4 pathway-dependent neutrophil responses. Frontiers in Immunology, 13, 888415.

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Isolation of Microglia from Mouse Brain

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Mice were deeply anaesthetized and then perfused intracardially with ice-cold DPBS (Mediatech 21-031CV). Cortex, striatum, and hippocampus were extracted and gently homogenized in staining buffer (HBSS (Life Technologies, 14175-095), 1% BSA, 1mM EDTA) on ice using a 2 ml polytetrafluoroethylene pestle (Wheaton), first in a 14 ml round-bottom tube (BD Falcon, 352059) and then in a 2 ml grinder chamber (Wheaton, 358029). Homogenates were filtered onto a 70 μm cell strainer (BD Falcon, 352350) and centrifuged for 10 min at 400g. Cell pellets were resuspended in 6 ml of 37% isotonic Percoll (Sigma, P4937) and then underlayed with 5 ml of 70% isotonic Percoll in a 15ml centrifuge tube (Corning, 430790). Tubes were then centrifuged at 600g for 40 min at 18°C, with no acceleration or decelaration. Cells at the 37–70% Percoll interface were recovered and washed once in 15 ml HBSS. Cell were then incubated in staining buffer on ice with CD16/CD32 (eBioscience, clone 93) antibody for 25 min, and then with CD11b-PE (BioLegend 101208, clone M1/70) and CD45-Alexa488 (BioLegend 103122, clone 3-F11) antibodies for 30 min. Cells were then washed once and filtered onto a 40 μm cell strainer (BD Falcon, 352340). Sorting was performed on a BD Influx cell sorter. Microglia were defined as singlets, CD11b⁺CD45^Low events, and emcompassed 90–95% of all CD11b⁺ events.

Crotti A., Benner C., Kerman B., Gosselin D., Lagier-Tourenne C., Zuccato C., Cattaneo E., Gage F.H., Cleveland D.W, & Glass C.K. (2014). Mutant Huntingtin promotes autonomous microglia activation via myeloid lineage-determining factors. Nature neuroscience, 17(4), 513-521.

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Microglia Enrichment and Immunophenotyping

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Hippocampi were collected and microglia were enriched by using density gradient separation and were prepared as described previously [12 (link), 65 (link)]. The cell suspension was then incubated with Fc receptor blocking antibody CD16/CD32 (1:200, BD Bioscience) and Fixable Viability Dye eFluor® 780 (1:1000, eBioscience) for 10 min at 4 °C. Subsequently, the following antibodies were used: anti-CD11b (1:200, clone M1/70, Biolegend), anti-CD45 (1:200, clone 30-F11, BioLegend), anti-CD11c (1:100, clone N418, Biolengend) and for lineage exclusion by a dump gate anti-CD3 (1:300, clone 17A2, Biolegend), anti-CD19 (1:300, clone 6D5, Biolegend), anti-CD45R (1:300, clone RA3-6B2, BD Bioscience), anti-Ly6C (1:300, clone AL-21, BD Bioscience) and anti-Ly6G (1:300, clone 1A8, BD Bioscience) and incubated for 30 min at 4 °C.

Mezö C., Dokalis N., Mossad O., Staszewski O., Neuber J., Yilmaz B., Schnepf D., de Agüero M.G., Ganal-Vonarburg S.C., Macpherson A.J., Meyer-Luehmann M., Staeheli P., Blank T., Prinz M, & Erny D. (2020). Different effects of constitutive and induced microbiota modulation on microglia in a mouse model of Alzheimer’s disease. Acta Neuropathologica Communications, 8, 119.

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Purification and Transfer of T-cell Subsets

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One week after the third recall, lymphocytes were collected from spleen of immunized or control group mice. Pelleted cells were resuspended in RobosepTM buffer (Stemcell technologies) at a final concentration of 10⁸ cells/mL. 5*10⁸ cells per group were purified by CD3+, CD8+ or CD4+ negative selection using an EasySep isolation kit (Stemcell Technologies). Cells were then injected intravenously (IV) in 4–5 week old C57BL/6 mice (10⁷ cell in 300μl). The remaining cells were marked with α-CD3-FITC (1:1000, clone 145-2C11, eBioscience), α-CD4-BrilliantViolet421 (1:500, clone M1/70, Biolegend) or α-CD8-PerCP (1:500, clone 53–6.7, Biolegend) stain and analyzed by FACS to assess the purity of the cell suspension, purity was always superior to 95%.

Castiglioni P., Hartley M.A., Rossi M., Prevel F., Desponds C., Utzschneider D.T., Eren R.O., Zangger H., Brunner L., Collin N., Zehn D., Kuhlmann F.M., Beverley S.M., Fasel N, & Ronet C. (2017). Exacerbated Leishmaniasis Caused by a Viral Endosymbiont can be Prevented by Immunization with Its Viral Capsid. PLoS Neglected Tropical Diseases, 11(1), e0005240.

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Immune Cell Profiling by Flow Cytometry

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The individual mouse immune cell populations were discriminated by antibody staining. FcR-blocking solution was applied for 10 min at 4 °C. After washing with 200 µl MACS buffer, staining against CD45 (BD Biosciences, clone 30-F11) and CD11b (Biolegend, clone M1/70) for total immune cells (CD45⁺), myeloid cells (CD45⁺ + CD11b⁺) and lymphoid cells (CD45⁺ + CD11b⁻) was performed. Furthermore, ectonucleotidases CD38 (BD Bioscience, clone 90/CD38), CD39 (BD Bioscience, clone Y23-1185) and CD73 (Biolegend, clone TY/11.8) were stained on the three main immune cell populations. Cells were stained for 20 min at 4 °C, followed by washing with 200 µl MACS buffer.

Massold T., Ibrahim F., Niemann V., Steckel B., Becker K., Schrader J., Stegbauer J., Temme S., Grandoch M., Flögel U, & Bouvain P. (2023). CD73 deficiency does not aggravate angiotensin II-induced aortic inflammation in mice. Scientific Reports, 13, 17125.

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Isolation and Characterization of Mouse Bone Marrow-Derived Macrophages

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Following treatments, BMDMs were harvested from cell culture plates using 5 mmol/l EDTA in PBS for 10 min at 37 °C. Cells were washed in FACS buffer (PBS containing 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich)). Cells were incubated with 10 μg/ml anti-mouse CD16/32 (clone 93, BioLegend) to block Fc receptors, then stained with anti-F4/80-BV421 (1:400; clone BM8, BioLegend) and anti-CD11b-PE (1:400; clone M1/70, BioLegend) in FACS buffer for 20 min at 4 °C. Stained cells were washed and resuspended in FACS buffer and acquired on a BD LSRFortessa (BD Biosciences) using FACSDiva software. Analysis was carried out using FlowJo software. For analysis, live cells were identified by gating based on forward and side scatter.

Phair I.R., Nisr R.B., Howden A.J., Sovakova M., Alqurashi N., Foretz M., Lamont D., Viollet B, & Rena G. (2022). AMPK integrates metabolite and kinase-based immunometabolic control in macrophages. Molecular Metabolism, 68, 101661.

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MSU Crystal-Induced Inflammation: Mechanisms and Resolution

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MSU crystals (3 mg) were injected intraperitoneally (i.p.) in mice and peritoneal exudates were collected at different time points (8, 24, 36 h) for the analysis of inflammatory cell composition. To examine the role of α_Mβ₂ integrin in MSU crystal-induced leukocyte recruitment, 50 μg of a blocking antibody against α_M integrin (clone M1/70; BioLegend) or an isotype control (clone RTK4530; BioLegend) were injected intravenously in mice 10 min prior to i.p. administration of the MSU-crystals. Mice were sacrificed and peritoneal exudates were analyzed 8 h after the MSU crystal injection.
The effect of DEL-1 on the regulation of resolution of MSU crystal-induced inflammation was investigated by injecting 5 μg of either DEL-1-Fc or Fc control i.p. 20 h after the treatment of mice with MSU crystals. 4 h after DEL-1-Fc injection, mice were sacrificed and clearance of apoptotic neutrophils was evaluated in peritoneal exudates.
In other experiments, 300 ng of RvD1 or vehicle control (PBS) were administered i.p. together with MSU crystals in wild-type and Del1^KO mice. 24 h after injection, the numbers of apoptotic neutrophils were measured in peritoneal exudates.

Kourtzelis I., Li X., Mitroulis I., Grosser D., Kajikawa T., Wang B., Grzybek M., von Renesse J., Czogalla A., Troullinaki M., Ferreira A., Doreth C., Ruppova K., Chen L.S., Hosur K., Lim J.H., Chung K.J., Grossklaus S., Tausche A.K., Joosten L.A., Moutsopoulos N.M., Wielockx B., Castrillo A., Korostoff J.M., Coskun Ü., Hajishengallis G, & Chavakis T. (2018). DEL-1 promotes macrophage efferocytosis and clearance of inflammation. Nature immunology, 20(1), 40-49.

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