Aortas were dissected, minced using scissors and enzymatically digested with 200 U/mL Liberase (Roche) and 40 U/mL DNase I (Sigma Aldrich) in HBSS plus 5% FCS for 1 h at 37°C. Cells were filtered through a 40 μm nylon strainer, washed with HBSS plus 5% FCS, collected by centrifugation at 400 g for 5 min at 4°C and then suspended in FACS buffer (PBS plus 0.2% FCS plus 1 mM EDTA). Murine Fc receptors were blocked using anti-CD16/32 antibodies (BioLegend) for 10 min on ice. Violet 510 Viability Dye (Cell Signaling Technology) was used to discriminate between live and dead cells. The cells were stained with the following antibodies for 30 min at 4°C: PerCP-Cy5.5-conjugated anti-CD45, APC-Cy7-conjugated anti-CD11b, FITC-conjugated anti-Ly6G, PE-Cy7-conjugated anti-F4/80, and APC-conjugated anti-Ly6C (all purchased from BioLegend). Flow cytometric analysis was performed using FACSCanto II flow cytometer (BD Biosciences) and DIVA Software (BD Biosciences).
Apc cy7 conjugated anti cd11b
Manufactured by BioLegend
Sourced in United States
APC-Cy7-conjugated anti-CD11b is a fluorescent-labeled antibody that binds to the CD11b cell surface antigen. CD11b is expressed on the surface of various immune cells such as monocytes, macrophages, and granulocytes. The APC-Cy7 fluorescent dye is used to label the anti-CD11b antibody, allowing for the detection and analysis of CD11b-expressing cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Apc conjugated anti ly6c, by BioLegend (3 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Percp cy5.5 conjugated anti cd45, by BioLegend (2 mentions)
Facs aria iiiu flow cytometer, by BD (2 mentions)
Dnase 1, by Merck Group (1 mentions)
Diva software, by BD (1 mentions)
Anti cd16 32 antibody, by BioLegend (1 mentions)
Liberase, by Roche (1 mentions)
Fitc conjugated anti ly6g, by BioLegend (1 mentions)
Pe cy7 conjugated anti f4 80, by BioLegend (1 mentions)
Anti cd45, by BioLegend (1 mentions)
Anti sca 1, by BioLegend (1 mentions)
Apc conjugated anti cd105, by BioLegend (1 mentions)
Pe conjugated anti cd44, by BioLegend (1 mentions)
Anti cd106, by BioLegend (1 mentions)
Facsverse system, by BD (1 mentions)
Pe cy7 conjugated anti cd45, by BioLegend (1 mentions)
Apc conjugated cd206 antibody, by BioLegend (1 mentions)
Fixation permeabilization solution, by BioLegend (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
7 protocols using apc cy7 conjugated anti cd11b
1
Murine Aortic Cell Isolation
2
Mesenchymal Stem Cell Expansion Kinetics
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AT-MSCs and CB-MSCs (2 × 104 cells) were seeded into 6-well plates on day 0. The total number of expanded cells were counted at day 1, 3 and 5. Cell doubling time (DT) was calculated by the following formula: DT = T × ln2/ln × (Xe/Xb), where T is the incubation time in any units; Xb is the cell number at the beginning of the incubation time; Xe is the cell number at the end of the incubation time.
Cultured cells were stained with FITC-conjugated anti-CD29 or anti-c-kit, PE-conjugated anti-CD44, anti-CD45, anti-CD106 or anti-Sca-1, APC-conjugated anti-CD105, and APC-Cy7-conjugated anti-CD11b (all from Biolegend, San Diego, CA, USA) at a concentration of 0.5 μg/mL for 30 min at 4 °C. The corresponding fluorophore-conjugated isotype controls were used for the gating of the positive-stained cells. Immunophenotypic analysis of 5000–10,000 cells of each sample was performed using FACSCanto II flow cytometer (BD Biosciences). The flow cytometry data were analyzed using Flowjo software (Tree Star, Ashland, OR, USA, ver. 10.0.7). Both assays were performed three times with duplicated samples.
Cultured cells were stained with FITC-conjugated anti-CD29 or anti-c-kit, PE-conjugated anti-CD44, anti-CD45, anti-CD106 or anti-Sca-1, APC-conjugated anti-CD105, and APC-Cy7-conjugated anti-CD11b (all from Biolegend, San Diego, CA, USA) at a concentration of 0.5 μg/mL for 30 min at 4 °C. The corresponding fluorophore-conjugated isotype controls were used for the gating of the positive-stained cells. Immunophenotypic analysis of 5000–10,000 cells of each sample was performed using FACSCanto II flow cytometer (BD Biosciences). The flow cytometry data were analyzed using Flowjo software (Tree Star, Ashland, OR, USA, ver. 10.0.7). Both assays were performed three times with duplicated samples.
3
Isolation and Analysis of Tissue-Resident Macrophages
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IVDs were harvested from young and aged mice (n = 5 each) and digested with 2 mg/ml collagenase type I solution (Product no. 032-22364, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) at 37°C overnight, before being passed through a nylon mesh filter with pore size 100 μm. The resulting single-cell suspensions were incubated with PE/Cy7-conjugated anti-CD45 (Clone: 30-F11, Product no. 103114, BioLegend, CA, USA) and APC-Cy7-conjugated anti-CD11b (Clone: M1/70, Product no. 101226, BioLegend) antibodies for 40 min at 4°C and subsequently treated with fixation/permeabilization solution (Product no. 420801, BioLegend). The cells were then incubated with APC-conjugated CD206 antibody (Clone: C068C2, Product no. 141708, BioLegend) for 30 min at 4°C and washed twice in wash buffer. The labeled cells were subjected to flow cytometry, in which 50,000 total events were acquired using a BD FACSVerse system (BD Biosciences, San Jose CA, USA), and the results were analyzed using FlowJo v10.7™ (Tree Star, Ashland OR, USA). Negative gates were determined using isotype control.
4
Flt3L-derived Dendritic Cell Generation and Modulation
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FL-DCs were prepared as previously described (37 (link)), with some modifications. In brief, bone marrow cells were cultured at a concentration of 3×105 cells/ml in RPMI-1640 medium containing 10% FBS supplemented with 200 ng/ml recombinant murine Flt3L (rmFlt3L, PeproTech, Rocky Hill, NJ, USA) and 2-mercaptoethanol (50 μM, Sigma-Aldrich), for 8 d. Cells were treated with MEHP, GW9662 (PPARγ antagonist; Tocris, Bristol, UK), or GW1929 (PPARγ agonist; Sigma-Aldrich) at various concentrations or with 0.1% ethanol (vehicle control) at the beginning of day 1 of culture. The medium containing rmFlt3L and/or chemicals or 0.1% ethanol was refreshed on days 4 and 6. On day 8, the cells were stained with the following antibodies, for phenotype analysis using flow cytometry: PE-conjugated anti-CD86 (GL-1; eBioscience), PerCP-cy5.5-conjugated anti-CD24 (M1/69; BioLegend), BV421-conjugated anti-CD45RA (14.8; BD Biosciences), APC-conjugated anti-CD11c (N418), APCcy7-conjugated anti-CD11b (M1/70; BioLegend), Alexa Fluor™ 700-conjugated anti-MHC II (M5/114.15.2), and LIVE/DEAD™ Fixable Red. For functional analysis, day-8 FL-DCs were washed and then stimulated with CpG1826 (10 μg/ml, InvivoGen, Carlsbad, CA, USA) for 24 h. The supernatants of FL-DCs were assessed for levels of cytokines using ELISA (R&D Systems).
5
Isolation and Flow Cytometry of Murine Blood, Bone Marrow, and Tissue Leukocytes
6
Monocyte Phenotyping by Flow Cytometry
7
Differentiation and Characterization of Murine Macrophages
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Bone marrow-derived macrophages from 9 to 12 weeks old ApoE–/– and ApoE–/–/Pad4–/– mice were prepared by flushing femurs and tibias followed by cell culture in RPMI medium supplemented with 20% FCS and 20 ng/mL murine M-CSF (Peprotech) for 7 days (26 (link)). To induce a M1- or M2a-like phenotype, macrophages (M0) were further incubated in the presence of 20 ng/mL IFN-γ (Peprotech) and 100 ng/mL LPS (Sigma Aldrich) or in medium supplemented with 20 ng/mL IL-4 (Peprotech), respectively. Macrophages’ phenotype was characterized by Real-time PCR and flow cytometry (FACS Canto II, BD). For flow cytometry, M0, M1-like and M2a-like macrophages were stained for CD11b, CD86 (M1 marker) and CD206 (M2 marker). The following antibodies were used: APC-Cy7-conjugated anti-CD11b, APC-conjugated anti-CD86 and Brilliant Violet 421-conjugated anti-CD206 (BioLegend).
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