After blocking Fc receptors, cells were incubated with appropriately diluted anti-mouse antibodies, including PE-F4/80 (BM8), PerCP-CD45 (30-F11), and APC-CD11b (M1/70) (Biolegend, San Diego, CA, USA). Appropriate species matched Abs served as isotype control. Acquisition of data was performed using a LSRII (BD Biosciences, Mountain View, CA, USA), and data analysis was conducted using the FlowJo software (Tree Star Inc., Ashland, OR, USA). Only live cells determined using LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies, Grand Island, NY, USA) were analyzed.
Apc cd11b m1 70
Manufactured by BioLegend
Sourced in United States
APC-CD11b (M1/70) is a fluorochrome-conjugated monoclonal antibody that binds to the CD11b cell surface antigen, also known as the integrin alpha M chain or Mac-1. CD11b is expressed on the surface of various immune cells, including monocytes, macrophages, and granulocytes. This antibody can be used in flow cytometry applications to identify and analyze these cell populations.
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Lab products found in correlation
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Rtk4530, by BioLegend (1 mentions)
Pe cd39 5f2, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Ly6 c pe cy7 hk1.4, by BioLegend (1 mentions)
Ly6g fitc 1a8, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd16 cd32, by BD (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
5 protocols using apc cd11b m1 70
1
Multicolor Flow Cytometry of Immune Cells
2
Multiparametric Flow Cytometry Analysis
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We incubated 1×106 cells with anti-mouse CD16/CD32 antibody [Becton, Dickinson, and Co. (BD) Biosciences, Franklin Lakes, NJ, USA USA] for 15 min at 4 ℃ to block Fc-receptors and avoid nonspecific binding. Cells were then stained for 20 minutes at 4 ℃ with a mix of fluorescent antibodies, including PacificBlue-CD3 (17A2, BioLegend, San Diego, CA, USA), PE/Cy7-NK1.1 (PK136, BioLegend), V500-CD8α (53-6.7, BioLegend), APC/Cy7-CD4 (RM4-5, BD Biosciences), FITC-CD44 (IM7, eBioscience, San Diego, CA, USA), FITC-Gr-1 (RB6-8C5, BD Biosciences), PerCP/Cy5.5-CD11c (N418, BioLegend), APC-CD11b (M1/70, BioLegend), BV510-B220 (RA3-6B2, BioLegend), and PerCP/Cy5.5-CD62L (MEL-14, BioLegend). Stained cells were detected using a FACSVerse Flow Cytometer (BD Biosciences). Flow data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
3
Bone Marrow-Derived Macrophage Isolation
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Macrophages were differentiated from BM cells as previously described (22 ). Briefly, BM cells were harvested by flushing the femurs with sterile PBS and then cultured in medium (RPMI1640 containing 10% FBS and 1× penicillin/streptomycin) supplemented with 10 ng/ml of murine macrophage colony-stimulating factor (M-csf; PeproTech) for 5 days at 37°C, 5% CO2. Medium was replaced on the second day of differentiation. BM-derived macrophages (BMDMs) were detached by using macrophage detachment solution (5 mM EDTA and 4 mg/ml lidocaine HCl in PBS) and by gentle scraping. Macrophages were characterized by FACS staining with APC Cd11b (M1/70, #17-0112-82, 1:100; BioLegend), FITC F4/80 (BM8, #11-4801-81, 1:100; eBioscience) and PE Cd39 (5F2, #12-3390-80, 1:100; eBioscience). APC rat IgG2b (RTK4530, #400611, 1:100; BioLegend), FITC rat IgG2b (eB149/10H5, #11-4031-81, 1:100; BioLegend), and PE mouse IgG1 (P3.6.2.8.1, 1:100; eBioscience) were used as isotype controls.
4
Multiparametric Flow Cytometry for MDSC Characterization
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Obtained single cell suspensions were stained with different panels of fluorescent labeled antibodies and analyzed by flow cytometry using the BD FACSCanto II (BD Biosciences) and the CytoFLEX (Beckman Coulter, Brea, California, USA). MDSCs were characterized using CD11b-APC (M1/70), Ly6G-FITC (1A8) and Ly6C-PE/Cy7 (HK1.4) antibodies (all from BioLegend, Figure 3 Supplementary Figures 4 5
5
Isolation and Characterization of Bone Marrow-Derived Macrophages
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Femurs and tibias were obtained from 6–10-week-old C57BL/6 mice, and their muscles were removed using protocols approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital (permit number 2018121710). Both ends of the bones were cut with scissors, and bone marrow cells were extruded with Dulbecco’s modified Eagle medium/F12 (GlutaMAX supplement, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) using a 25-gauge needle. Bone marrow cells were cultured in complete macrophage medium supplemented with recombinant macrophage colony-stimulating factor (ProSpec-Tany TechnoGene Ltd., Rehovot, Israel). After 7 days of culture, nonadherent cells were eliminated, and adherent cells were harvested for assays. Phenotypic characterization of BMDMs identified as F4/80+/CD11b+/CD11c- was assayed using flow cytometry (BD FACSVerse; BD Biosciences, Franklin Lakes, NJ, USA) (47 (link)). Macrophages were incubated with rat anti-mouse CD16/CD32 (BD Biosciences) to block the Fc receptors. The cells were then stained using the following anti-mouse antibodies: CD11b-APC (M1/70, BioLegend, San Diego, USA), F4/80-Brilliant Violet 420 (BM8, BioLegend), and CD11c-Alexa Fluor 488 (N-418, STEMCELL Technologies, Vancouver, Canada). Data were analyzed using FlowJo software (version 8.8.6, TreeStar, Ashland, OR).
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