Single-cell suspensions from pooled quadriceps and TA muscles were generated as previously described.84 The interrogation of live macrophage populations was performed via staining with Zombie NIR viability dye (Cat. #423105; BioLegend, San Diego, CA, USA), CD11b (PerCP-Cy5, Cat. #101228; BioLegend), F4/80 (PE, Cat. #123110; BioLegend), Siglec-F (BV421, Cat# 56268; BD Biosciences, San Jose, CA, USA), Ly6C (FITC, Cat. #128006; BioLegend), CD206 (Alexa Fluor 647, Cat. #141711; BioLegend). Cells were analyzed using a BD FACSAria Fusion flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, version 10; FlowJo, Ashland, OR, USA).
Cd206 alexa fluor 647
Manufactured by BioLegend
Sourced in United States
CD206-Alexa Fluor 647 is a fluorescent-labeled antibody that binds to the CD206 receptor, also known as the macrophage mannose receptor. It is a membrane-bound C-type lectin that is primarily expressed on the surface of macrophages and dendritic cells. This product can be used for flow cytometry and other immunological applications to identify and characterize cells expressing the CD206 receptor.
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Lab products found in correlation
Fc block, by BD (3 mentions)
F4 80 alexa fluor 488, by BioLegend (2 mentions)
Cd11c phycoerythrin, by BioLegend (2 mentions)
Cd11b percp cy5, by BioLegend (2 mentions)
F4 80 pe, by BioLegend (2 mentions)
Cd16 32 fcγriii fcγrii, by BD (2 mentions)
Cd16.2 fcγriv, by BioLegend (2 mentions)
Novocyte 2060, by Agilent Technologies (2 mentions)
Facsaria fusion flow cytometer, by BD (1 mentions)
Ly6c fitc, by BioLegend (1 mentions)
Siglec f, by BD (1 mentions)
Cd11c fitc, by BD (1 mentions)
F4 80 pe, by Abcam (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
100 mm cell strainer, by BD (1 mentions)
Cd19 fitc, by BioLegend (1 mentions)
Be0089, by BioXCell (1 mentions)
Cd4 alexa fluor 647, by BioLegend (1 mentions)
Nk 1.1 alexafluor 647, by BioLegend (1 mentions)
9 protocols using cd206 alexa fluor 647
1
Macrophage Phenotyping by Flow Cytometry
2
Isolation and Analysis of Stromal Vascular Fraction
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The SVF was isolated from inguinal subcutaneous or epidydimal visceral fat depot of male FGF21KO and WT mice after rmFGF21 treatment or fat transplantation. The mice were anesthetized and the fat pads were removed and digested in 0.1% (w/v) collagenase type I (Invitrogen) for 30 min at 37 °C with gentle shaking. The digestion mixture was passed through a 100 mm cell strainer (BD Biosciences) and centrifuged at 800×g for 10 min at 4 °C. The SVF pellets were collected and washed twice, 1 × 105 freshly isolated cells were triple stained with F4/80-PE (Abcam, ab105156, clone CI:A3-1, 1:50), cd206-Alexa Fluor 647 (Biolegend, 141712, clone C068C2, 1:100) and cd11c-FITC (BD Biosciences, BD 557400, clone HL3, 1:100) on ice for 30 min in dark. Species-matched IgG was used as non-specific isotype controls. After washing, the cells were analyzed with LSR Fortessa Cell Analyzer (BD Biosciences).
3
Characterization of Immune Cell Populations
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Therapeutic anti-PD-1 (BE0146) and isotype control antibody (BE0089) were produced by BioXcell. Antibodies used for flow cytometry were purchased from eBioscience (Anti-mouse CD3ε FITC, CD11b eFluor 660) and Biolegend (Anti-mouse CD8a Alexa Fluor® 647, NK 1.1 Alexa Fluor® 647, Ly-6G/Ly-6C (Gr-1) Alexa Fluor® 488, CD19 FITC, CD45R/B220 Alexa Fluor® 647, F4/80 Alexa Fluor® 488, CD206 Alexa Fluor® 647, FOXP3 Alexa Fluor® 488, CD4 Alexa Fluor® 647, CD11c Alexa Fluor® 488, I-A/I-E Alexa Fluor® 647, CD16/32).
4
Macrophage Phenotyping by Flow Cytometry
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Peritoneum macrophages were stimulated with serumfree medium supplemented with LPS (5 µg/mL). After stimulation for 12 h, cells were suspended in staining buffer (eBioscience, # 00-4222) and stained with F4/80-FITC (#11-4801) (ebioscience), CD11b-PE (#12-0112-82) (eBioscience), and CD206-Alexa Fluor 647 (#141712) (BioLegend, San Diego, CA, USA) for 30 min in darkness at 4℃. Stained cells were washed twice in staining buffer and finally suspended in 1% paraformaldehyde in PBS. Flow cytometry data were acquired and analyzed using a FACS Calibur (BD Biosciences, San Diego, CA, USA).
5
Liver Macrophage Phenotyping in Mice
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Liver tissues from male C57BL/6J mice were minced and digested for 30 min at 37 °C with type II collagenase (Sigma-Aldrich) in PBS containing 2% BSA (pH 7.4). The cell suspension was filtered and then spun at 3000 rpm for 5 min to separate the floating liver tissue fraction from the stromal vascular cell (SVC) pellet. The SVCs were resuspended in PBS supplemented with 2% FBS and incubated with Fc-Block (BD Bioscience).
To determine macrophage phenotype fluorescence-activated cell sorting analysis FACSAriaII (BD Bioscience) was performed using following antibodies, NK1.1-PerCP Cy5.5 (eBioscience, San Diego, CA), CD3-PerCP Cy5.5 (eBioscience, San Diego, CA), CD19-PerCP Cy5.5(eBioscience), TER119-PerCP Cy5.5 (eBioscience, San Diego, CA), CD45-APC-Cy7(eBioscience, San Diego, CA), Gr-1Fluor450 Ly-6G(PB) (eBioscience, San Diego, CA), F4/80-PE-Cy7(Bio Legend, San Diego, CA), CD11b-PETR (Invitrogen, Carlsbad, CA, USA), CD11c-PE(eBioscience, San Diego, CA), CD206 Alexa Fluor 647 (Bio Legend, San Diego, CA). Data analysis and compensation were performed using FlowJo (Tree Star).
To determine macrophage phenotype fluorescence-activated cell sorting analysis FACSAriaII (BD Bioscience) was performed using following antibodies, NK1.1-PerCP Cy5.5 (eBioscience, San Diego, CA), CD3-PerCP Cy5.5 (eBioscience, San Diego, CA), CD19-PerCP Cy5.5(eBioscience), TER119-PerCP Cy5.5 (eBioscience, San Diego, CA), CD45-APC-Cy7(eBioscience, San Diego, CA), Gr-1Fluor450 Ly-6G(PB) (eBioscience, San Diego, CA), F4/80-PE-Cy7(Bio Legend, San Diego, CA), CD11b-PETR (Invitrogen, Carlsbad, CA, USA), CD11c-PE(eBioscience, San Diego, CA), CD206 Alexa Fluor 647 (Bio Legend, San Diego, CA). Data analysis and compensation were performed using FlowJo (Tree Star).
6
Multicolor Flow Cytometry Panel
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Fc Block (20 m g/mL; BD Biosciences) was used to block cell-surface antigens. After blocking cells were stained with fluorophore conjugated antibodies or isotype control antibodies. Fluorophore-conjugated primary antibodies were purchased from BioLegend: F4/80-Alexa Fluor 488, CD11b-PerCP/Cy5.5, CD11c-phycoerythrin, and CD206-Alexa Fluor 647. After incubation with antibodies, cells were washed and centrifuged, re-suspended in a washing buffer, and analyzed on a FACSCalibur using FlowJo 10.0.6.
7
Isolation and Characterization of Wound Macrophages
8
Macrophage Polarization Analysis in Brain Tissue
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To study macrophage polarization, leukocytes were isolated from freshly harvested brain tissue following previously published protocol [83 ]. The protocol was slightly modified and is included in the supplementary information. As previously, Fc receptors were blocked with antibodies against CD16/32 FcγRIII/FcγRII (BD Bioscience, USA; clone, 2.4G2) and CD16.2 FcγRIV (Biolegend, USA; clone, 9E9) and then stained with PE-Cy7 CD45 (BioLegend, USA; clone, I3/2.3), PE F4/80 (BioLegend, USA; clone, BM8), Alexa Fluor 488 CD86 (BioLegend, USA; clone, GL-1), and Alexa Fluor 647 CD206 (BioLegend, USA; clone, C068C2). Macrophages were defined as CD45high, F4/80+. Cells were run in a Novocyte 2060 (ACEA Biosciences, USA) at a rate of 14 μL/min.
9
Multiparameter Flow Cytometry of MSCs and Macrophages
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For MSC cell-surface characterization, the following antibodies were used: APC-Vio770 CD29 (Miltenyi Biotec; clone, HMβ1-1), VioBright FITC CD44 (Miltenyi Biotec, USA; clone, REA665), PerCP Sca-1 (Biolegend, USA; clone, D7), PE CD31 (Miltenyi Biotec, USA; clone, 390), PE CD45 (Biolegend, USA; clone, 30-F11), and PE-Vio770 CD105 (Miltenyi Biotec, USA; clone, MJ7/19). For analysis of in vitro macrophages, we first blocked Fc receptors with CD16/32 FcγRIII/FcγRII (BD Bioscience, USA; clone, 2.4G2) and CD16.2 FcγRIV (Biolegend, USA; clone, 9E9) and then stained with PE CD45 (Biolegend, USA; clone, 30-F11), Alexa Fluor 647 CD206 (BioLegend, USA; clone, C068C2), and PE CD163 (ThermoFisher, USA; clone, TNKUPJ). A Novocyte 2060 (ACEA Biosciences, USA) was used to measure mean fluorescence intensity of GFP from transfected MSCs, MSC cell-surface markers, and macrophage markers. Data was analyzed in FlowJo software (BD Biosciences, USA).
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