Macrophages cultured for 7 days were carefully harvested using Dispase enzyme solution and gentle scraping. Cells were counted and a minimum of 3 × 105 cells were used for each FACS staining. Fc receptors were blocked in 3% BSA in 1× HBSS (Gibco) for 10 min. As this study focused on membrane proteins, permeabilization and fixation of cells were omitted. Instead, cells were re-suspended in PBS and incubated with the respective antibodies for 30 min. Antibodies used for FACS were αCD163-APC and αCD90-PE (both Biolegend), CD11b-V450, CD11c-PE, CD40-FITC, CD45-PE, CD209-Cy5.5, CD80-V450, CD86-V450, CD146-PE, and CD206-FITC (all BD Pharmingen). Unstained cells were used as control, as well as an IgG control. Cells were washed twice after antibody incubation and re-suspended in PBS for counting. Cell sorting was performed on a LSR-II instrument (BD Bioscience) using FACSDiva8 acquisition and analysis software. Cells were gated in three steps; first, cells were discriminated by size employing forward and side scatter (FSC and SSC, respectively). Second, doublets were removed by pulse-geometry gating the area and height of FSC. Lastly, the fluorescence signal of cells positive for respective markers was gated directly, plotting it against the SSC area.
Cd206 fitc
Manufactured by BD
Sourced in United States
CD206-FITC is a fluorescently-labeled antibody that binds to the CD206 receptor, also known as the mannose receptor, which is expressed on the surface of certain immune cells, such as macrophages. This product is used in flow cytometry and other laboratory techniques to identify and quantify cells expressing the CD206 receptor.
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Lab products found in correlation
Cd11b pe, by BD (3 mentions)
Cd14 apc, by BD (3 mentions)
Cd90 pe, by BD (3 mentions)
Cd45 pe, by BD (2 mentions)
Cd4 pe cy7, by BD (2 mentions)
Facsdiva 8, by BD (2 mentions)
Cd31 fitc, by BD (2 mentions)
Cd44 apc h7, by BD (2 mentions)
Facsverse system, by BD (2 mentions)
Cd45 fitc, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Cd86 v450, by BD (1 mentions)
Cd146 pe, by BD (1 mentions)
Cd80 v450, by BD (1 mentions)
Cd40 fitc, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Lsrii instrument, by BD (1 mentions)
Cd11c pe, by BD (1 mentions)
Cd163 fitc, by BioLegend (1 mentions)
Cd33 pe, by Thermo Fisher Scientific (1 mentions)
12 protocols using cd206 fitc
1
Multicolor Flow Cytometry of Macrophages
2
Macrophage Phenotype Characterization
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Cells were detached using Cell Dissociation Solution Non-enzymatic (Sigma-Aldrich) and stained with fluorochrome-conjugated antibodies against CD163-FITC, CD11b-APC (both from BioLegend), CD206-FITC (BD BioSciences), MSR1-Alexa Fluor® 700 (R&D Systems, MN, USA) and CD33-PE (eBioscience) at 5 and 20 days after M-CSF or M-CSF/RANKL stimulation. Cells (50 × 103) were acquired using a Gallios Flow Cytometer (Beckman Coulter, Pasadena, CA, USA) and analyzed by FlowJo software (Tree Star, Inc., Ashland, OR, USA).
3
Flow Cytometry Analysis of Renal Cells
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Kidneys were collected from sacrificed animals for flow cytometry analysis following the standard manufacturer's protocol. Briefly, kidneys were reperfused, excised, and incubated in collagenase IV for 30 min. Then, monocytes were separated by Percoll (Sigma, St. Louis) gradient. We analyzed the renal cells by multicolor flow cytometry. The monoclonal antibodies used were F4/80 PerCP, CD11b PE, CD206 FITC, MIG PE, IL10 APC, p40 (IL12/IL23) PE‐Cy7, CCR7 PerCP‐Cy5.5, IFNγ FITC, CD45.1 Pacific Blue, CD45.2 APC‐Cy7, GM‐CSF PE, MHC II IAB PerCP, and CD36 FITC (all purchased from BD Biosciences, Franklin Lakes). Samples were acquired on a FACSCanto using FACSDIVA software (BD Biosciences), followed by analysis with FLOWJO software (Tree Star, San Carlo, CA). Fluorescence voltages were determined using matched unstained cells. Compensation was performed using cells (BD Biosciences) single‐stained with CD3 PErCP, CD4 FITC, CD8 APC‐CY7, CD4 PE‐CY7, CD4 PerCP‐Cy5.5, CD3 PE, or CD3 APC. Samples were acquired up to at least 200,000 events in a live mononuclear gate, followed by a doublets exclusion gate, following the standard manufacturer's procedure. The compensation process was performed according to the “fluorescence minus one” method.
4
Phenotypic Characterization of Macrophages
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Flow cytometric assay was performed using FACSCantoII Analyzer (BD Biosciences, USA). Differentiated macrophages were detached with trypsin and washed with 1× PBS. For the detection of macrophage surface markers, cells were incubated with monoclonal mouse anti-human antibodies CD163-Cy5.5, CD206-FITC, and CD80-FITC (BD Biosciences, USA), or relevant isotypes (BD Biosciences) for 1 h at 4 °C in dark. After washing with 1× PBS, the fluorescence was compared to isotypes with 10,0000 events recorded. All flow cytometric data were analyzed using Flowjo software, version 10 (Tree Star, USA).
5
Multiparameter Flow Cytometry Characterization
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The characterization of differentiated MDM was performed via flow cytometry with the following mAbs: CD11b BV 786 (Biolegend, San Diego, CA, USA; clone IRCF44), CD14 APC (BD Biosciences, Franklin Lakes, NJ, USA; clone M5E2), CD206 FITC (BD Biosciences, clone 19.2), HLA-DR BV711 APC (BD Biosciences, clone G46-6), CD16 PE (BD Biosciences, clone 3G8), CD4 BV 421 (BD Biosciences, clone RPA-T4), CD8FITC (BD Biosciences, clone RPA-T8), CD19 APC (BD Biosciences, cloneHIB19) and CD69PECy7 (Biolegend, clone G10F5). Live dead staining (ThermoFisher, Waltham, MA, USA) was performed as a control for vitality. Labelled cells were fixed in PBS−/− 1× 1% formalin. A minimum of 50.000 events were acquired for each sample using a BD Lyric (BD Biosciences) and analyzed by FACS Diva 8.0.1 (BD Biosciences).
6
Multiparametric Immune Cell Profiling
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Passage 2 cells were suspended in Hank’s Balanced Salt Solution (HBSS) at a density of 5 × 105 cells/mL and stained for 30 min on ice with the following antibodies: CD11b-PE, CD11c-PE-Cy7, CD14-APC, CD31-FITC, CD44-APCH7, CD45-FITC, CD73-BV421, CD90-PE, CD105-PerCP-Cy5.5, CD206-FITC, and HLADR-APC (BD, Franklin Lakes, NJ). Cell surface antigens were analyzed using a triple-laser FACS Verse™ system (BD).
7
Polarization of THP1 and Mouse Myeloid Cells
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THP1 cells were pretreated with 50 ng/mL PMA (Sigma) for 48 hours, then stimulated with macrophage M2 polarization inducers IL4 and IL13 (both at 40 ng/mL, Peprotech) for 48 hours. THP1 or whole blood cells from mice were resuspended in Stain Buffer (BD, 554656) for 15 minutes. The THP1 cells were stained with CD11b-APC (BD, 550019), CD64-FITC (BD, 555527), CD86-FITC (BD, 557343), and CD206-FITC (BD, 551135). The whole blood cells were stained with Ly6C-PE/Cy7 (BioLegend, 128017), Ly6G-APC (BioLegend, 127613), CD11b-FITC (BioLegend, 101205), CD86-PE (BioLegend, 105106), and CD206-PerCP/Cy5.5 (BioLegend, 141716). The cells were detected on a CytoFLEX (Beckman Coulter). The results were analyzed by FlowJo v10.0.
8
Immunophenotypic Analysis of hAdMSCs
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Analyses of the immunophenotype of hAdMSCs were conducted as previously described [19 (link), 20 (link)]. After being washed, hAdMSCs were subjected to incubation for half an hour at 4 °C with primary antibodies against human, including CD34-PE (cat#550761, BD Biosciences), CD44-PE (cat# 559942, BD Biosciences), CD29-PE (cat#556049, BD Biosciences), CD73-PE (cat# 550257, BD Biosciences), CD90-PE (cat# 555596, BD Biosciences), CD45-PE (cat# 560975, BD Biosciences), CD106-PE (cat# 561679, BD Biosciences), CD105-PE (cat# 560839, BD Biosciences), CD206-FITC (cat#5 51135, BD Biosciences) and HLA-DR-PE(cat# 555561, BD Biosciences). Following washing, hAdMSCs were subjected to incubation with secondary antibodies for half an hour at 4 °C. MSCs were then examined using FACSCalibur (BD Biosciences) and the FlowJo program (FlowJo, LCC). Moreover, adipogenic and osteogenic differentiation media were used to cultivate the cells. We performed oil red O staining to assess the adipogenic differentiation of cells (Solarbio, China). Alkaline phosphatase (ALP), an early osteogenic marker, and Alizarin Red (ARS), a late osteogenic marker, were used to quantify the osteogenic differentiation of the cells.
9
Comprehensive Immune Cell Profiling
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Single cell suspensions were incubated with anti-mouse monoclonal antibodies CD8α PE-CF594 (C53-6.7), XCR1 PE (ZET), CD11c BV421 (N418), MHC II APC-Cy7 (M5/114), CD103 BV510 (N290), CD326 PE-Cy7 (G8.8), CD4 PE-Cy7 (RM4-5), CD11b PERCP-Cy5-5 (M1/70), CD206 FITC (C068C2), CD301b APC (URA-1), Siglec-H APC (551), PDCA-1 BV650 (927), CD274 FITC (10F.9G2), TLR3 PE (11F8), TLR5 APC (ACT5), and TLR7 PE (A94B10), purchased from BD Biosciences or BioLegend. For intracellular cytokine staining of TLR3 and TLR7 cells were first fixed with 1% paraformaldehyde (Sigma-Aldrich) and then incubated with relevant antibodies to intracellular targets in a buffer containing 0.2% saponin to permeabilize cells (Sigma-Aldrich). Post intracellular staining, cells were washed and kept in serum free buffer until analysis. Multi-parameter analysis was performed on a LSRFortessa (BD Biosciences). All data were analyzed with FlowJo (v10, Tree Star, Ashland, OR, USA).
10
Apoptosis and Cytokine Profiling of MSCs and THP1 Cells
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The percentage of apoptotic MSCs were evaluated using a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Pharmingen) according to the manufacturer’s instructions. MSCs were collected and underwent incubation with PI and annexin V-FITC at 37 °C for 15 min away from the light.
THP1 cells were acquired to incubate with primary antibody against anti-human CD14-FITC (BD bioscience, USA), CD206-APC (BD bioscience, USA), IL-10-efluor 660 (eBioscience, USA), and TNF-α-PE (Biolegend, USA) at 4 °C for 30 min, protected from the light. TNF-α and IL10 cytokine staining were conducted according to the intracellular cytokine staining protocol. Murine BALF cells were cultured for an additional 24 hours after collection and stained with anti-mouse F4/80-BV421, TNF-α-PE, CD206-FITC antibodies (BD bioscience, USA). Flow cytometry assay was conducted with a BD LSR II flow cytometer. Data were analyzed by the Flowjo7.6 software (Becton Dickinson).
THP1 cells were acquired to incubate with primary antibody against anti-human CD14-FITC (BD bioscience, USA), CD206-APC (BD bioscience, USA), IL-10-efluor 660 (eBioscience, USA), and TNF-α-PE (Biolegend, USA) at 4 °C for 30 min, protected from the light. TNF-α and IL10 cytokine staining were conducted according to the intracellular cytokine staining protocol. Murine BALF cells were cultured for an additional 24 hours after collection and stained with anti-mouse F4/80-BV421, TNF-α-PE, CD206-FITC antibodies (BD bioscience, USA). Flow cytometry assay was conducted with a BD LSR II flow cytometer. Data were analyzed by the Flowjo7.6 software (Becton Dickinson).
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