One day after IR, hearts were perfused with pre‐cold PBS. Left ventricle tissues subjected to IR injury were removed and dissociated with gentleMACS dissociator (Miltenyi Biotec, USA). The digestion was performed in 5 ml HBSS buffer contained Collagenase II (Worthington, 1.5 mg/ml), Collagenase IV (Worthington, 1.5 mg/ml) and DNase I (Sigma, 60U/ml) at 37℃ for 30 min at a speed of 200 rpm. The resulting suspension was filtered (70 μm) to generate a single‐cell suspension. The suspension was centrifuged at 300 × g for 5 min. Cardiac cells were then resuspended in DMEM media (supplemented with 10% FBS and 1% Penicillin‐Streptomycin) and cultured for 2 h (37°C, 5% CO2). Cells were then washed with PBS and cardiac macrophages were enriched in the adherent cells (de Couto et al., 2017 ). Cells were collected and incubated with flow cytometry antibodies, including FITC‐CD4, PE‐F4/80, PerCP‐Cy5.5‐CD86 and PE‐Cy7‐CD206 (all from BioLegend, USA) at 4°C in the dark for 15 min. After PBS washing, the phenotype of cardiac macrophages was detected by BD FACSCanto II flow cytometer (BD Bioscience, USA).
Cd206 pe cy7
Manufactured by BioLegend
Sourced in United States
CD206-PE-Cy7 is a fluorescently-labeled antibody that binds to the CD206 antigen. CD206, also known as the mannose receptor, is a cell surface receptor expressed on macrophages and dendritic cells. The PE-Cy7 conjugate provides a fluorescent signal that can be detected using flow cytometry or other fluorescence-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd11c apc efluor780, by Thermo Fisher Scientific (4 mentions)
F4 80 pe, by Thermo Fisher Scientific (4 mentions)
Cd11b bv510, by BioLegend (3 mentions)
Gr1 apc cy7, by BioLegend (3 mentions)
Cd4 fitc, by BioLegend (3 mentions)
Cd11b apc, by Thermo Fisher Scientific (3 mentions)
Cd4 pe, by BioLegend (3 mentions)
Cd86 apc, by BioLegend (3 mentions)
F4 80 pe, by BioLegend (2 mentions)
Murine trustain fcx, by BioLegend (2 mentions)
Alexafluor700 f4 80, by Bio-Rad (2 mentions)
Percp cy5.5 cd11c, by BD (2 mentions)
Siglecf pe cf594, by BD (2 mentions)
Cd54 apc, by BioLegend (2 mentions)
Pb cd40, by BioLegend (2 mentions)
Bv711 cd64, by BioLegend (2 mentions)
Bv605 ia ie, by BD (2 mentions)
Buv737 cd43, by BD (2 mentions)
Cd71 pe, by BioLegend (2 mentions)
Cd11c apc, by BioLegend (2 mentions)
25 protocols using cd206 pe cy7
1
Cardiac Macrophage Isolation and Analysis
2
Cardiac Leukocyte Isolation Procedure
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Cardiac leukocyte isolation was performed as previously described (Eberhardt et al., 2019 (link)). Briefly, hearts were perfused with phosphate-buffered saline (PBS) and disaggregated mechanically and enzymatically with 0.2% trypsin solution (Gibco). The digested tissue was pressed through a 70 μm cell strainer (BD Falcon), and cells were isolated by 35 and 70% bilayer Percoll (GE Healthcare) density gradient centrifugation. Viable cell numbers were determined by trypan blue dye exclusion using a Neubauer chamber, and absolute cell number was obtained corresponding to the whole heart. Cells were stained with the following antibodies: anti-mouse fluorescein isothiocyanate (FITC)-CD3, APC-Cy7-CD4, PE-Cy7-CD8, PE-CD19, PerCP-Cy5.5-CD11b, FITC or APC-Cy7-CD11c, PE-F4/80, PE-Cy7-CD206, APC-Ly6G, and APC-Cy7-Ly6C (all from BioLegend). Stained samples were acquired using FACS Canto I and II cytometers (Becton Dickinson), and the data were analyzed using FlowJo software (Tree Star). Non-specific fluorescence was determined using isotype controls.
3
Multicolor Flow Cytometry Panel
4
Monocyte-derived Macrophage Phenotyping
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Bone marrow cells were flushed from femurs and tibias, centrifuged, then exposed to geys buffer for 5 min on ice, incubated with Fc block (Murine TruStain FcX, Biolegend, 1/50 dilution) for 15 min, incubated with antibodies for 20 min at 4 °C, and washed before sorting CD115+/CD11c-/NK- monocytes using an Influx cell sorter (BD) [68 (link)]. Monocytes were cultured in complete RPMI medium (Thermo Fisher Scientific) and exposed to 100 ng/mL CSF1, 50 µM Q-VD-OPh and 50 µM Ac-YVAD-cmk. After five days, macrophages were studied by flow cytometry using AlexaFluor700-F4/80 (#MCA497A700, Bio-Rad), BV510-CD11b (#101245, Biolegend), PerCP/Cy5.5-CD11c (#560584, BD), APC/Cy7-GR1 (#108424, Biolegend), PE-CF594-SiglecF (#562757, BD), PE-CD71 (#113808, Biolegend), PE/Cy7-CD206 (#141720, Biolegend), APC-CD54 (#116120, Biolegend), PB-CD40 (#124626, Biolegend), BV711-CD64 (#139311, Biolegend), BV605-IA-IE (#563413, BD), BUV737-CD43 (#564398, BD). For the sorting of monocytes, the antibodies used were the following: APC-CD11b (#17-0112-83, Thermo Fisher Scientific), PE/Cy7-CD11c (#561022, BD), FITC-NK (#553164, BD), PerCP/Cy5.5-Ly6C (#560525, BD), AlexaFluor700-Ly6G (#561236, BD), biotin-CD115 (#135507, Biolegend), PE-Streptavidin (#554061, BD). The data were analyzed with FlowJo software v. 10.0.00003.
5
Interstitial Macrophages Identification
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Right lungs were digested with the Lung Dissociation kit (Miltenyi Biotec), filtered, erythrocytes were removed using ACK and nucleated cells were collected. Cells were washed with ice-cold PBS, incubated with Fc block (Murine TruStain FcX, Biolegend, 1/50 dilution) for 15 min, incubated with the antibodies for 20 min at 4 °C, washed, then fluorescence was measured with a BD LSRFortessa X-20. Antibodies used were the following: AlexaFluor700-F4/80 (#MCA497A700, Bio-Rad), FITC-CD45 (#103108, Biolegend), BV510-CD11b (#101245, Biolegend), PerCP/Cy5.5-CD11c (#560584, BD), APC/Cy7-GR1 (#108424, Biolegend), PE-CF594-SiglecF (#562757, BD), PE-CD71 (#113808, Biolegend), PE/Cy7-CD206 (#141720, Biolegend), APC-CD54 (#116120, Biolegend), PB-CD40 (#124626, Biolegend), BV711-CD64 (#139311, Biolegend), BV605-IA-IE (#563413, BD), BV650-CD24 (#563545, BD), BUV737-CD43 (#564398, BD). The data were analyzed with FlowJo software v. 10.0.00003. Interstitial macrophages were selected according to their larger size (FSC) and granularity (SSC), on CD45 expression to select leukocytes and GR1-positive cells will be excluded to remove neutrophils, they are equally CD11b high, SiglecF negative, IA-IE positive and CD24 negative.
6
Comprehensive Monocyte Phenotyping by Flow Cytometry
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Phenotyping of monocytes and monocyte‐derived macrophages was performed by flow cytometry using fluorochrome‐conjugated monoclonal antibodies specific for HLA‐DR (FITC, BD Biosciences Franklin Lakes, NJ, USA), CD14 (Pacific Orange, Thermo Fisher Scientific), CD16 (BUV737, BD), CD64 (APC‐Cy7, Biolegend, San Diego, CA, USA), CD86 (BV711, BD), CD206 (PE‐Cy7, Biolegend) and FRβ (APC, Biolegend). The expression of the GM‐CSF receptor (BV650, BD) and the M‐CSF receptor (PE‐Cy7, Biolegend) on monocyte subsets was analysed by a separate flow cytometry panel (including the aforementioned CD14, CD16 and HLA‐DR antibodies). Cells were measured on LSR‐II (BD) flow cytometer. For comparison of the mean fluorescence intensity between experiments, the LSR‐II flow cytometer was calibrated for each run using FACSDiva CS&T research beads (BD). Data were analysed using Kaluza software (BD). Monocytes and macrophages were gated by FSC/SSC, doublets were excluded, and dead cells were excluded using Zombie dye (Biolegend). To exclude contaminating lymphocytes in the monocyte gate, cells negative for both HLA‐DR and CD14 were gated out. Monocyte subsets were gated based on CD14 and CD16 expression.6 Gating strategy plots are available as Supplementary figure 6 .
7
Isolation and characterization of immune cells from the brain
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Immune cells were isolated from whole-brain homogenates as recently described (15 (link)). Briefly, brain tissue was processed using the neural tissue dissociation kit (Miltenyi Biotec) in accordance with the manufacturer’s specifications. Afterward, cells were separated using a discontinuous Percoll density gradient (30–37–70% Percoll layers), which results in the enrichment of all immune cell types and removes a lot of myelin, which is auto-fluorescent. Following density gradient centrifugation, immune cells were washed and stained for 30 min at 4 °C using the following antibodies: CD11b APC-Cy7 (BD Biosciences), CD45.2 Pacific blue, CD86 PE-Cy5, and CD206 PE-Cy7 (BioLegend). Respective isotype control antibodies or unstained samples were used to determine positive populations. Myelin debris and dead cells were excluded by FSC/SSC gating, and singlet populations were analyzed.
For flow cytometric analyses of blood samples, EDTA-blood was stained at 4 °C for 15 min with the following antibodies: B220 Pacific Blue, CD3 AF700, CSF-1R BV605 (BioLegend), and CD11b APC-Cy7 (BD Biosciences). For more details see Waltl et al. (15 (link)).
Flow cytometry was performed using an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).
For flow cytometric analyses of blood samples, EDTA-blood was stained at 4 °C for 15 min with the following antibodies: B220 Pacific Blue, CD3 AF700, CSF-1R BV605 (BioLegend), and CD11b APC-Cy7 (BD Biosciences). For more details see Waltl et al. (15 (link)).
Flow cytometry was performed using an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).
8
Multiparametric Phenotyping of Lung Immune Cells
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Pleural and Bronchoalveolar cells were preincubated with murine Fc block CD16/CD32 and then stained with the following rat anti-mouse antibodies: anti-F4/80-APC (1:200; eBioscience, clone BM8), anti-SiglecF-PE (1:200, BD Bioscience, clone E50-2440), anti-Ly6G-V450 (1:200, eBioscience, clone 1A8), anti-CD4-PE (1:200; BD Bioscience, clone RM4-5) and anti-CD19-APC (1:200; BD Bioscience, clone 1D3). Macrophages were further analysed using anti-CD169-FITC (1:200, Biolegend, clone 3D6.112) and CD206-PE-Cy7 (1:200, Biolegend clone C068C2). Fluorescence Minus One (FMO) controls were used for each group with a pool of cells of all groups. The samples were run on a FACSVerse flow cytometer (BD Biosciences) and analysed using FACSuite software. Doublets and debris were excluded. CD169 and CD206 expression is expressed as mean fluorescence intensity (MFI) normalized by FMO (MFI-FMO).
9
Multiparametric Flow Cytometry of tPCLS
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Single‐cell suspensions of tPCLS were generated as described above. Single‐cell suspensions were blocked with FcR blocking reagent (Miltenyi Biotec) in 0.5% PBS‐BSA for 20 min, stained with fluorochrome‐conjugated antibodies, and analyzed on a FACSSymphony A5SE flow cytometer (BD Biosciences). Live single cells were identified by FSC and SSC characteristics. The data were analyzed using FlowJo V10 (TreeStar). All antibodies and secondary reagents were titrated to determine optimal concentrations. Comp‐Beads (BD) were used for single‐color compensation to create multicolor compensation matrices. For gating, fluorescence minus one control was used. The instrument calibration was controlled daily using Cytometer Setup and Tracking Beads (BD Biosciences). The following antibodies were used: CD3‐BUV805 (#612896, BD Biosciences), CD4‐BB630 (#562316, BD Biosciences), CD8‐BV650 (#743067, BD Biosciences), CD14‐PerCP‐Cy5.5 (#561116, BD Biosciences), CD15‐BUV805 (#742057, BD Biosciences), CD16‐BV650 (#563692, BD Biosciences), CD19‐APC‐H7 (#560252, BD Biosciences), CD25‐PE‐Cy7 (#557741, BD Biosciences), CD33‐BV510 (#563257, BD Biosciences), CD45‐AF700 (#368514, BD Biosciences), CD80‐BV711 (#740801, BD Biosciences), CD206‐PE/Cy7 (#321124, BioLegend), CD326‐FITC (#324203, BioLegend), HLA‐DR‐APC/Fire750 (#307658, BioLegend), MerTK‐BV421 (#367603, BioLegend).
10
Dissociation and Flow Cytometry Analysis of Renal Tumors
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Tumors and adjacent healthy renal tissues were dissociated using the human Tumor Dissociation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany, 130-095-929) and GentleMACS System (Miltenyi Biotec). Samples were acquired with a LSRII/Fortessa flow cytometer (BD, Heidelberg, Germany) expressed as mean fluorescence intensity (MFI). CompBeads (BD) were used for single color compensation to create multi-color compensation matrices. For gating, fluorescence minus one (FMO) controls were used. Prior to experiments, all antibodies and secondary reagents were titrated to determine optimal concentrations.
For staining of FPN, extracellular staining of patient-derived single cell suspensions was performed, containing CD33 BV510 (BD, 563257), MerTK BV421 (Biolegend, San Diego, CA, USA, 367603), CD45 AF700 (Biolegend, 368513), CD 64 BV605 (Biolegend, 305033), CD206 PE-Cy7 (Biolegend, 321124), CD326 PE-CF594 (BD, 565399), HLA-DR APC-Cy7 (Biolegend, 307658), and FPN PE (Novus, Wiesbaden, Germany, NBP1-21502).
For staining of FPN, extracellular staining of patient-derived single cell suspensions was performed, containing CD33 BV510 (BD, 563257), MerTK BV421 (Biolegend, San Diego, CA, USA, 367603), CD45 AF700 (Biolegend, 368513), CD 64 BV605 (Biolegend, 305033), CD206 PE-Cy7 (Biolegend, 321124), CD326 PE-CF594 (BD, 565399), HLA-DR APC-Cy7 (Biolegend, 307658), and FPN PE (Novus, Wiesbaden, Germany, NBP1-21502).
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