To examine macrophage phenotypic changes in the BALF, macrophages from BALF were separated via magnetic-activated cell sorting (MACS) using magnetic beads. For flow cytometry analysis, the macrophages after EVAloe treatment were fixed, permeabilized, and stained with anti-human CD11b-Percp/Cyanine5.5 (BioLegend, Cat. No. 301327, clone ICRF44) and M1 macrophage marker (anti-human CD80-PE, BioLegend, Cat. No. 305207, clone 2D10) and M2 macrophage marker (anti-human CD206-APC monoclonal Abs, BioLegend, Cat. No. 321109, clone 15-2) for flow analysis. All data were analyzed using FlowJo.
Pe anti human cd80
Manufactured by BioLegend
Sourced in United States
PE anti-human CD80 is a flow cytometry antibody that binds to the CD80 cell surface protein, also known as B7-1, on human cells. CD80 is a costimulatory molecule expressed on antigen-presenting cells that plays a key role in T cell activation and immune regulation.
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Lab products found in correlation
Apc anti human cd11b, by BioLegend (3 mentions)
Pe anti mouse cd86, by BD (2 mentions)
Pe cy7 anti human fcεriα mab cra1 clone aer 37, by Thermo Fisher Scientific (2 mentions)
Pe anti mouse ige rme 1, by BioLegend (2 mentions)
Alexa fluor 647 anti mouse cd8α, by BioLegend (2 mentions)
Alexa fluor 647 anti mouse cd4, by BioLegend (2 mentions)
Anti cd117 apc, by BioLegend (2 mentions)
Pe anti mouse fcεriα mab mar1, by BioLegend (2 mentions)
Pe anti mouse ifn γ clone xmg1, by BioLegend (2 mentions)
Apc anti mouse il 4 clone 11b11, by Thermo Fisher Scientific (2 mentions)
Apc anti human cd19 clone hib19, by BioLegend (2 mentions)
Pe cy7 anti human ige clone mhe 18, by BioLegend (2 mentions)
Pe anti human cd203, by BioLegend (2 mentions)
Alexa fluor anti human cd83 clone hb15e, by BioLegend (2 mentions)
Apc anti human cd1a, by BD (2 mentions)
Mouse fc block, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Anti cd11c apc, by BioLegend (2 mentions)
Pe anti human cd86, by BioLegend (2 mentions)
11 protocols using pe anti human cd80
1
Macrophage Phenotypic Analysis in BALF
2
Phenotyping M1 and M2 Macrophages
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Flow cytometry was performed using standard protocols. An Fc receptor block (BioLegend) was incubated with samples to reduce nonspecific antibody binding for half an hour at 4 °C. Afterward, the cells were incubated for half an hour at 4 °C with fluorochrome-tagged monoclonal antibodies from BioLegend, such as anti-human CD11b APC (cat# 301330), anti-human CD80 PE (cat# 305220), anti-human CD11c APC (cat# 301614), and anti-human CD206 FITC (cat#321110). Cell populations were gated as follows: M1 macrophages (CD11b+CD 80+) and M2 macrophages (CD11b+ CD206+). We performed flow cytometry using a FACSCalibur flow cytometer (BD Bioscience), and FlowJo software (FlowJo, LCC) was used for data analysis.
3
Macrophage Phenotype Analysis by Flow Cytometry
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The tMACs or iMACs were stimulated with LPS and IFN-γ for the indicated time. The single-cell suspensions were then prepared and incubated with an antibody or antibody cocktails for 15 min at room temperature for cell surface staining. Antibodies used in this study were PE Mouse IgG1, κ isotype (Biolegend, Cat: 400113, Clone: MOPC-21, Lot: B245984), APC Mouse IgG1, κ isotype (Biolegend, Cat: 400119, Clone: MOPC-21, Lot: B243042), FITC Mouse IgG1, κ isotype (Biolegend, Cat: 400107, Clone: MOPC-21, Lot: B199152), APC anti-human CD206 (Biolegend, Cat: 321109, Clone: 15-2, Lot: B348965), APC anti-human CD86 (Biolegend, Cat: 305411, Clone: IT2.2, Lot: B351349), PE anti-human CD80 (Biolegend, Cat:305208, Clone: 2D10, Lot: B330518), PE anti-human CD163 (Biolegend, Cat: 333606, Clone: GHI/61, Lot: B347256), FITC anti-human CD14 (Biolegend, Cat: 325604, Clone: HCD14, Lot: B268830) and APC anti-humanCD11B (Biolegend, Cat: 301309, Clone: ICRF44, Lot: B278346). These antibodies were used at a 1:100 dilution. Data were recorded on Beckman DxFLEX and analyzed with the FlowJo V10 software.
4
Multiparameter Flow Cytometry Analysis
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Single cell suspensions were pretreated with mouse Fc-Block™ (BD Biosciences/ Pharmingen) and incubated with fluorophore-labeled antibodies or appropriate isotype controls. Acquisitions were performed using a BD FACSCanto™ II (BD Bioscience). For intracellular staining of cytokines BD Cytofix/Cytoperm (BD Biosciences) was used. Analysis was done using BD FACSDiva™ and FlowJo (Treestar INC.) software. Following antibodies were used: Phycoerythrin-(PE), allophycocyanin-(APC) and PE/Cy7 anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or BioLegend), PE-anti-mouse IgE (RME-1, Biolegend), APC-anti-CD11c, Alexa Fluor® 647 anti-mouse CD8α and Alexa Fluor® 647 anti-mouse CD4 from Biolegend (San Diego, CA), PE-anti-mouse CD86 (BD Pharmingen), APC-anti- CD117 (Biolegend), and PE- anti-mouse FcεRIα mAb (Mar1, Biolegend). Intracellular cytokine stain included PE anti-mouse IFN-γ (clone XMG1.2, Biolegend) and APC anti-mouse IL-4 (clone 11B11, eBioscience). Anti-human CD1c (BDCA-1, clone AD5-8E7, Miltenyi Biotech), APC-anti-human CD19 (clone HIB19, BioLegend), PE/Cy7 anti- human IgE (clone MHE-18, BioLegend), PE anti-human CD203 (Biolegend), Alexa Fluor® anti human CD83 (clone HB15e), PE anti-human CD80 (Biolegend) and APC anti-human CD1a (BD Pharmingen).
5
Western Blot and Flow Cytometry Antibodies
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Primary antibodies used for western blotting included anti‐human Alix (1:1000, mouse monoclonal, Cell Signalling, #2171S), anti‐human TSG101 (C‐2) (1:1000, mouse monoclonal, Santa Cruz Biotechnology, #sc‐7964), anti‐human CD9 (1:1000, rabbit monoclonal, Cell Signalling, #13403), anti‐human Ago2 (1:1000, rabbit polyclonal, Abcam, #ab32381). Secondary antibodies used for western blot included: anti‐mouse horseradish peroxidase‐linked antibody (1:3000, sheep, GE Healthcare Life Sciences, #NA931V), and anti‐rabbit horseradish peroxidase‐linked antibody (1:4000, donkey, GE Healthcare Life Sciences, #NA934V). Antibodies used for flow cytometry experiments included PE/Cyanine 7 anti‐human CD11c (1:200, BioLegend, #337216), PE anti‐human CD80 (1:100, BioLegend, #305208), PerCP/Cyanine5.5 anti‐human CD86 (1:200, BioLegend, #305420), Brilliant Violet 421™ anti‐human CD274 (B7‐H1, PD‐L1) (1:50, BioLegend, #329714), FITC anti‐human HLA‐DR (MHCII) clone: REA805 (1:200, Miltenyi, #130‐111‐788). In all experiments particle‐free sterile commercial Phosphate‐buffered saline (PBS) pH 7.2 was used (Thermo Fisher Scientific, Gibco™ #20012027).
6
Macrophage Polarization Markers Analysis
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The polarization-related surface markers of RAW264.7, MH-S, and THP-1 macrophages were detected by flow cytometry. In our previous study (5 (link)), we found that 10 μg/ml CSE-EVs could significantly promote M1 polarization of RAW264.7 macrophages, thus we treated macrophages (5×104 cells/cm2) with 10 μg/mL EVs in EVs-free RPMI 1640 mediums for 24 h in this study. For positive control, 2 μM SF1670 and 1 μM AG14361 were used to inhibit PTEN and PAPR1 expression of THP-1 cells, respectively. Then, the cells were collected and stained with the following antibodies on ice for 20 min in the dark. APC anti-mouse CD86, APC anti-mouse CD80, APC anti-human CD11b, PE anti-human CD86, and PE anti-human CD80 (Biolegend, San Diego, CA, USA) were used in this procedure. After being washed twice with PBS, cells were fixed with fixation buffer and then analyzed by flow cytometry (CytoFLEX, Beckman).
7
Characterization of Mature Monocyte-Derived Dendritic Cells
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Sensitive conjugated antibodies were used to determine the target population of mature moDCs. Cells were removed from the culture plate and washed from the medium with PBS. The moDCs were then stained with conjugated antibodies: Pacific Blue™ anti-human CD3 (#300330, Biolegend, USA), Pacific Blue™ anti-human CD19 (#302232, Biolegend, USA), Pacific Blue™ anti-human CD56 (#362520, Biolegend, USA), Pacific Blue™ anti-human CD20 (#302328, Biolegend, USA), PerCP/Cyanine5.5 anti-human CD11c (#301624, Biolegend, USA), FITC antibody to human HLA-DR (#307604, Biolegend, USA), APC anti-human CD64 (#305014, Biolegend, USA), PE anti-human CD80 (#305208, Biolegend, USA), PE anti-human CD83 (#305308, Biolegend, USA) according to the manufacturer’s instructions. Data were analyzed using FACS Aria III (BD Biosciences, USA) and BD FACSDivaTM software version 7.0.
8
Polarizing and Characterizing Macrophages
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MDMs were cultured in 12-well plates and exposed to an M1 polarization stimulus using E. coli lipopolysaccharide (LPS) (Sigma, 20 ng/ml) plus recombinant human interferon (rh-IFN)-γ (Fisher, 20 ng/ml) and into M2a using rhIL-4 (20 ng/ml) for 48 h per prior methods (2 (link), 27 (link)). Resident cells without any stimulation were used for baseline comparison. Aliquots incubated in human Fc-γ Block (Biolegend, 422301) for 30 min on ice and then stained using phycoerythrin (PE) anti-human CD80 (Biolegend, 305208, Clone 2D10) and phycoerythrin cyanine tandem conjugate (PE/Cy7) anti-human CD11b (Biolegend, 101216, Clone M1/70) or allophycocyanine (APC) anti-human CD163 (Biolegend, 326510, Clone RM3/1) and allophycocyanin cyanine 7 tandem conjugate (APC/Cy7) anti-human CD206 (Biolegend, 321120, Clone 15-2). Next, FITC anti-human CD68 (Biolegend, 333806, Clone Y1/82A) intracellular staining was performed post-fixation and permeabilization according to the manufacture's recommendations. Stained cells were gated via viable CD11b+subsets and identified by CD68CD80 (M1) and CD163CD206 (M2) markers using FlowJo (Tree Star).
9
Multiparameter Flow Cytometry Analysis
10
Differentiation of Dendritic Cells from PBMCs
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PBMCs were collected from healthy volunteers and cultured in AIM-V medium (A3021002, Gibco) at 37°C and 5% CO2. Two hours later, cell flasks were gently shaken to suspend and remove the unattached and semi-attached cells, which were cultured to induce immature DCs (iDCs) in AIM-V medium supplemented with 800U/ml GM-CSF (215-GMP-050, R&D Systems) and 500U/ml IL-4 (204-GMP-050, R&D Systems) for 6 days. The supernatant of iDCs was mixed with fresh medium in a ratio of 1:1 to induce mature DCs (mDCs) for 16–18 h with 800U/ml GM-CSF, 500U/ml IL-4, 160ng/ml IL-6 (206-GMP-050, R&D Systems), 5ng/ml IL-1β (201-GMP-100, R&D Systems), 5ng/ml TNFα (210-GMP-100, R&D Systems), and 1μg/ml PGE2 (2296/10, Tocris). The iDCs and mDCs were identified by flow cytometry using PE-anti-human CD80 (305208, BioLegend), PE-anti-human CD83 (305308, BioLegend), PE-anti-human CD86 (305406, BioLegend), PE-anti-human CD14 (367104, BioLegend), PE-anti-human CD11c (301606, BioLegend), PE-anti-human HLA-DR (307606, BioLegend), PE-anti-human HLA-ABC (311406, BioLegend), and PE-anti-human CCR7 (353204, BioLegend) antibodies.
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