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Clone m1 70

Manufactured by Thermo Fisher Scientific
Sourced in United States

The Clone M1/70 is a monoclonal antibody that recognizes the CD11b antigen. CD11b is a cell surface integrin expressed on a variety of immune cells, including monocytes, macrophages, and neutrophils. The Clone M1/70 antibody can be used for the identification and analysis of CD11b-positive cells in research applications.

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15 protocols using clone m1 70

1

Flow Cytometry and Immunohistochemistry Antibody Sources

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Sources of the antibodies for flow cytometry analysis are listed as follow: mouse Fc Block (1:100; 553142; BD Biosciences), mouse CD45 (1:100; clone 30-F11; Biolegend), mouse Gr1 (1:100; clone RB6-8C5; Biolegend), mouse CD11b (1:100; clone M1/70; eBiosciences), mouse CD11c (1:100; clone N418; Biolegend), mouse F4/80 (1:100; clone MB8; Biolegend), mouse Ly6G (1:100; clone 1A8; Biolegend), mouse Ly6C (1:100; clone HK1.4; Biolegend), human ENTPD2 (1:25; PA5-26333; Sigma-Aldrich), mouse Entpd2 (1:25; ab150503; Abcam), human ENTPD1 (1:100; clone A1; Biolegend), and mouse Entpd1 (1:100; clone Duha59; Biolegend). Sources of the antibodies for immunohistochemistry are listed as follow: human ENTPD2 (1:200; ab150503; Abcam), human GLUT1 (1:1000; ab15309Abcam), and human CA9 (1:500; ab1508L; Abcam).
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2

Multicolor Flow Cytometry Analysis of Pancreatic Immune Cells

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Pancreatic cells isolated from each group were washed by a fresh FACS buffer for three times, then cell suspensions were treated with the anti-mouse CD16/CD32 (1:100 ratio, clone 93, eBioscience, Santa Clara, CA, USA) for 15 min to block the Fc receptor. Cell suspensions were subsequently labeled with anti-mouse PE-Cyanine 5 conjugated CD11b (1:500 ratio, clone M1/70, eBioscience, Santa Clara, CA, USA) and anti-mouse FITC conjugated F4/80 (1:500 ratio, clone BM8, eBioscience, Santa Clara, CA, USA), and/or anti-mouse PE conjugated Ly6G (1:500 ratio, clone RB6-8C5, eBioscience, Santa Clara, CA, USA) for 30 min on ice in the dark to detect macrophages or neutrophils. Stained cells were measured with the FACS Calibur (Becton Dickinson, San Jose, CA, USA) and flow cytometric data were analyzed using CellQuestPro software.
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3

Quantifying Microglial Amyloid Uptake

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Mice were injected intraperitoneally with methoxy‐XO4 (Tocris) at 10 mg/kg bodyweight in a DMSO/PBS mixture at 1:10 ratio as described previously with slight modifications (Heneka et al, 2013). Hippocampi were isolated 3 h after methoxy‐XO4 injection and processed into single‐cell suspension with a potter. The homogenate was filtered through a cell strainer (70 μm) and was separated by 37% Percoll gradient centrifugation at 800 g for 30 min at 4°C. The myeloid containing phase was collected and washed once with PBS. Fc receptor blocking antibody CD16/CD32 (1:200, clone 2.4G2, BD Bioscience) was applied in order to prevent unspecific binding, and dead cells were stained using the Fixable Viability Dye eFluor® 780 (1:1,000, eBioscience) at 4°C for 20 min. Cells were washed once and then stained with primary antibodies directed against CD11b (1:200, clone M1/70, eBioscience), CD45 (1:200, clone 30‐F11, eBioscience), and CD36 (1:200, clone 72‐1, eBioscience) at 4°C for 20 min. Cells were washed again, and then, frequencies of viable methoxy‐XO4+ CD11b+CD45low microglia cells were determined by flow cytometry using a FACS Canto II (BD Biosciences) and analyzed using FlowJo (Tree Star). WT mice injected with methoxy‐XO4 were used as controls to determine the methoxy‐XO4 threshold for non‐phagocytosing cells. Corresponding isotype control antibodies were used.
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4

Isolation of Pdgfrb-eGFP Embryonic Mesenteric Cells

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Mesenteries of E14 Pdgfrb-eGFP embryos were dissected and dissociated at 37°C and 550 rpm for 5-10 min with 2 mg/ml Collagenase IV (Life Technologies) and 0.2 mg/ml DNase I (Roche) in PBS supplemented with 0.2% fetal bovine serum (FBS; Gibco). Digests were quenched by adding 2 mM EDTA, filtered through a 70 µm nylon filter (BD Biosciences) and washed twice with FACS buffer (0.5% FBS and 2 mM EDTA in PBS). Digests were incubated for 15 min with 5 µg/ml rat anti-mouse CD16/CD32 IgG (clone 93, eBioscience) to block Fc-receptor binding and subsequently stained with rat anti-PECAM1/CD31-PE-Cy7 (0.67 µg/ml clone 390, eBioscience), hamster anti-PDPN-eF660 (2 µg/ml clone eBio8.1.1., eBioscience), rat anti-CD11b-eF450 (4 µg/ml clone M1/70, eBioscience) and rat anti-CD45-eF450 (4 µg/ml clone 30-F11, eBioscience) antibodies for 30 min on ice. Sytox Blue (1 mM; Life Technologies) was used to assess cell viability. Single stained samples were used for compensation. Samples were analyzed on a BD Cytoflex S flow cytometer equipped with CytExpert software (BD Biosciences) and processed using FlowJo 10.3 software (FlowJo, LLC).
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5

Immunofluorescence Staining of Cultured Cells

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Cell cultures were fixed in 4% paraformaldehyde (pH 7.4) for 15-20 min at RT and blocked in 10% normal goat serum with 0.1% Triton-X-100 for 1 hr. Cells were then incubated with primary antibody overnight at 4°C and in secondary antibodies for 1 hr at RT. Cells were counterstained with DAPI for 5 min at RT. Primary antibodies used were rabbit anti-mouse Olig2 (1:200; cat. no. AB9610, Millipore), rat anti-mouse MBP (1:200; clone 12, Millipore), rat anti-mouse Ki67 (1:200; clone SoIA15, eBioscience), rabbit anti-mouse GFAP (1:200; cat. no. Z0334, Dako) and rat anti-mouse CD11b (1:200; clone M1/70, eBioscience) and secondary antibodies used were goat anti-rabbit AF488 (1:1000; cat. no. A-11008) and goat anti-rat AF594 (1:1000; cat. no. A-11007; both Life Technologies). For live/dead staining unfixed cells were stained using the LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Life Technologies). Immunofluorescence was detected using an EVOS microscope at 10X magnification, and n = 6 wells per condition, with one mean value from multiple images calculated for each well.
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6

Immunofluorescence Staining of Cultured Cells

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Cell cultures were fixed in 4% paraformaldehyde (pH 7.4) for 15-20 min at RT and blocked in 10% normal goat serum with 0.1% Triton-X-100 for 1 hr. Cells were then incubated with primary antibody overnight at 4°C and in secondary antibodies for 1 hr at RT. Cells were counterstained with DAPI for 5 min at RT. Primary antibodies used were rabbit anti-mouse Olig2 (1:200; cat. no. AB9610, Millipore), rat anti-mouse MBP (1:200; clone 12, Millipore), rat anti-mouse Ki67 (1:200; clone SoIA15, eBioscience), rabbit anti-mouse GFAP (1:200; cat. no. Z0334, Dako) and rat anti-mouse CD11b (1:200; clone M1/70, eBioscience) and secondary antibodies used were goat anti-rabbit AF488 (1:1000; cat. no. A-11008) and goat anti-rat AF594 (1:1000; cat. no. A-11007; both Life Technologies). For live/dead staining unfixed cells were stained using the LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Life Technologies). Immunofluorescence was detected using an EVOS microscope at 10X magnification, and n = 6 wells per condition, with one mean value from multiple images calculated for each well.
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7

Multicolor Flow Cytometry Analyses

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Single-cell suspensions were washed in PBS containing 5 mM EDTA and 0.5% BSA. The following antibodies were used for analysis: anti-CD11c (hamster; clone N418; eBioscience); anti-MHCII [I-A] (rat; clone NIMR-4; eBioscience); anti-CD11b (rat; clone M1/70; eBioscience); anti-CD103 (hamster; clone 2E7; BioLegend); anti–PD-L2 (rat; clone TY25; eBioscience); anti-FoxP3 (rat; clone FJK-16s; eBioscience); anti-IFNγ (rat; clone XMG1.2; eBioscience); anti-Vβ5 (mouse; clone MR9-4; BD); and anti-CD86 (rat; clone GL-1; BioLegend). Staining for intracellular FoxP3 and IFNγ expression was performed using FoxP3/TF staining buffer kit (#00-5523; eBioscience). Staining for ALDH activity was performed as previously reported using Aldefluor reagent kit (#01700; STEMCELL Technologies). Flow cytometry data were analyzed using Flowjo software.
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8

Isolation of CD11b+/Ly-6G+ Cells from Metastatic Livers

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Seven days or 14 days following splenic injection of the breast cancer cells, CD11b+/Ly-6G+ cells were isolated from the metastasis-bearing livers. Livers were first perfused with perfusion medium (Gibco, Waltham, MA, USA, cat#17701-038) for 10 minutes at 5 ml/min. Following perfusion, livers were minced and dissociated for 1 h in a mix of collagenase A, collagenase B and hyaluronidase (2 mg/ml each, Roche, Basel, Switzerland, cat#11088793001 and 11088031001; Sigma-Aldrich, St Louis, MO, USA, cat#H3884).
To purify CD11b+/Ly-6G+ cells [34 (link),35 (link)], cells were blocked in 2.4G2 (1:10 dilution) for 30 minutes and then incubated for 30 minutes, in the dark, with an antibody cocktail containing anti-mouse CD11b-APC (1:10000 dilution, eBioscience, clone M1/70, cat#17-0112-82) and rat anti-mouse Ly-6G-PerCP-Cy5.5 (1:1000 dilution, BD Biosciences, San Jose, CA, USA, clone 1A8, cat#560602). Subsequently, cells were washed once with PBS and then incubated with Live/Dead stain (1:1000 dilution) for 30 minutes. Cells were then washed once with PBS and resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS complemented with 2% fetal bovine serum (FBS)). Samples were read on the BD FACS Canto (BD Biosciences).
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9

Comprehensive Immune Cell Profiling by Flow Cytometry

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PEC, MLN and spleen cells were incubated in the presence of PMA (50ng/ml), ionomycin (500ng/ml) and Brefeldin A (5μg/ml) (Sigma) for 4 h at 37°C, before surface staining with fluorochrome-conjugated antibodies against CD3 (BD, clone 500A2), γδTCR (Biolegend, clone GL3), CD4 (DB, clone RM4-5), CD8 (eBioscience clone 53-6-7), CD11b (Biolegend, clone M1/70), F4/80 (eBioscience, clone BM8) Ly6G (BD, RB6-8C5), MHCII (eBioscience, clone M5/114.15.2), CD44 (eBioscience, clone IM7), CD62L (eBioscience, clone MEL-14). Cells were fixed and permeabilised using the Dako IntraStain Fixation and Permeabilization Kit, before intracellular staining with fluorochrome-conjugated antibodies against IL-17A (eBioscience, clone 17B7) and IFNγ (eBioscience, clone XMG1.2). Flow cytometric data was acquired with a BD FACSCanto II and analysed using FlowJo software (Tree Star, Inc.). Gates are set on respective Fluorescence Minus One (FMOs). To investigate reactive oxygen species (ROS) activity within phagocytes, 123-dihydrorhodamine assays were carried out as previously described [83 (link)].
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10

Hepatic Macrophage Isolation and Characterization

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For hepatic macrophage isolation, sections of liver were placed in digestion buffer with 0.25 mg mL−1 collagenase type I, 1.2 mM CaCl2, 0.1 mg mL−1 DNase I in PBS for a minimum of 20 min, filtered through 100 μm strainer, and spun at 50 × g to pellet hepatocytes. Supernatant was collected and spun at 800 × g to pellet nonparenchymal cells. Cells were resuspended in histodenz (20%) and gradients generated using a 1380 × g spin for 15 min with no brake. Cells at the interface were collected, washed, and stained for CD45-APC (1:50 dilution, clone 30-F11, ThermoFisher #17-0451-82), F4/80-PE (1:50 dilution, clone BM8, ThermoFisher #12-4801-82) and CD11b-FITC (1:50 dilution, clone M1/70, ThermoFisher #11-0112-41). Cells were flow sorted at the University of Colorado Cancer Center Flow Cytometry Shared Resource using the MoFlo XDP70 (Beckman Coulter, Indianapolis, IN). Resident macrophages were defined as CD45+/F4/80+Hi/ CD11b+Lo and recruited macrophages were defined as CD45+/F4/80+Lo/ CD11b+Hi70 (link) (gating strategy, Supplementary Fig. 4).
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