Bone marrow-derived macrophages were transduced with a control vector (pMX-FLAG-IRES-EGFP) or the constitutively active form of STAT5A (pMX-FLAG-IRES-EGFP-STAT5A1*6), differentiated to osteoclasts in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL), and collected. The collected cells were stained with PE-anti-mouse CD80 (BD PharmingenTM, San Diego, CA, USA), PE-anti-mouse CD 86 (BD PharmingenTM), PE-anti-mouse MHC class II (eBioscience, San Diego, CA, USA), and APC-anti-mouse CD11c (eBioscience) for 20 min at 4 °C. Stained cells were analyzed using a Navios flow cytometer with Kaluza software (Beckman Coulter, Brea, CA, USA).
Pe anti mouse cd86
Manufactured by BD
Sourced in United States
PE anti-mouse CD86 is a fluorochrome-conjugated antibody that binds to the CD86 cell surface antigen expressed on mouse cells. It can be used for the identification and quantification of cells expressing CD86 in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Pe anti human cd80, by BioLegend (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)
Apc anti mouse cd206, by BioLegend (2 mentions)
Eflour 450 anti mouse cd11b, by Thermo Fisher Scientific (2 mentions)
10 protocols using pe anti mouse cd86
1
Osteoclast Differentiation and Characterization
2
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).
3
Multiparameter Flow Cytometry Analysis
4
Flow Cytometry Analysis of Immune Cell Subsets
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samples were seeded in 6 cm dish and induced according to the experimental protocol. Pre-prepared cells were further incubated with the corresponding fluorochrome-labelled primary antibody at 25 °c for 30 min. Sony ID7000 Spectral Cell Analyzer was used for flow cytometry analyses. Data were analysed and presented using FlowJo 10.8.1. Antibodies for flow cytometry are listed here: FITC anti-mouse/human CD11b (BioLegend, 101206, clone: M1/70), PE anti-mouse CD206 (BioLegend, 141705, clone: C068C2), BB515 anti-mouse CD8 (BD Pharmingen, 564422), PE anti-mouse CD86 (BD Pharmingen, 553692), PERCP anti-mouse/human CD11b (BD Pharmingen, 566416), APC anti-mouse CD206 (BD Pharmingen, 565250), APC-CY7 anti-mouse FSV780 (BD Pharmingen, 565388), BV421 anti-mouse CD3 (BD Pharmingen, 562600), BV510 anti-mouse CD45 (BD Pharmingen, 563891), BV421 anti-mouse F4/80 (BD Pharmingen, 565411), FITC AnnexinV (BD Pharmingen, 556420) and Fixable Viability Stain 780 (BD Pharmingen, 565388).
5
Flow Cytometry Characterization of Macrophage Subsets
6
Characterization of Tumor-Associated Macrophages
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Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in Hank's Balanced Salt Solution (HBSS) (Gibco) for 15 min at RT, then blocked in 100 μL of FACS Buffer (HBSS without calcium, 2% FBS and 1mM EDTA) with 1% bovine serum albumin (BSA) (Sigma) for 20 min at 4°C. Cells were washed with FACS buffer between each step. Cells suspensions of 50 μL were incubated for 15 min at RT with 0.5 μL Mouse TruStain FcX (Biolegend, 101319) to prevent nonspecific Fc receptor binding. Samples were immediately stained with the following primary antibodies for 30 min at 4°C: 0.125μg/100μL eFlour 450 anti-mouse CD11b (Thermo Fisher Scientific, Clone M1/70), 0.5μg/100μL FITC anti-mouse F4/80 (Thermo Fisher Scientific, Clone BM8), 0.5μg/100μL PE anti-mouse CD86 (BD Biosciences Clone GL1), and 0.5μg/100μL APC anti-mouse CD206 (BioLegend, Clone C068C2). Cells were washed 2x with FACS buffer and analyzed using a Guava EasyCyte 12HT benchtop flow cytometer (MilliporeSigma). Flow cytometry plots were analyzed using FlowJo v10.7.1 software. Macrophages were characterized as CD11b+ F4/80+ populations. Within the gated macrophage population, M1/M2 gates were made using a control sample for each tumor, stained only for CD11b and F4/80 in the absence of M1/M2 markers to account for background fluorescence. M1 macrophages were characterized as CD86+ while M2 macrophages were CD206+.
7
Cytofluorimetric Analysis of RAW Cells
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Cytofluorimetric assessments were conducted from RAW cells cultured under different conditions. This was done in 6-well plates (Costar, Corning Incorporated), seeding an initial quantity of 5 x 105 cells/well. RAW cells were harvested and cell suspensions were washed with PBS 1X FBS 2% and then labeled with Live/Dead (1:1000) (Live/Dead Fixable Near-IR Dead Cell Stain Kit, Invitrogen by Thermo Fisher Scientific) for 30 minutes at 4°C in the dark. Cells were washed with PBS 1X FBS 2% and stained with the following antibodies: anti-mouse CD80 APC (MACS Miltenyi Biotech); anti-mouse CD86 PE (BD Biosciences Pharmingen). After washing, cells were fixed with 1% formalin 15 minutes at room temperature. Cells were examined using a FACSCanto flow cytometer (BD) and data were analyzed using FlowJo software (TreeStar). Plotted ratio was calculated between the percentage value of RAW cells with positive M2 phenotype for the expression of CD80 and CD86, respectively (see gating strategy described in S1 Fig in S1 File ), divided by the percentage value of untreated RAW cells with positive M2 phenotype without Poly(I:C) and/or CSE treatment.
8
Macrophage Activation Profiling
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First, the peptides M2pep and scM2pep were labeled with Streptavidin-Alexa 633. In parallel, the activated macrophages were harvested by Accutase (Sigma-Aldrich) treatment. Afterwards, both M1 and M2 macrophages were incubated with 20 μM of labeled peptide for 30 min at 4°C. Prior to flow cytometry analysis, the cells were stained with anti-mouse CD206-FITC (Biolegend) and anti-mouse CD86-PE (BD Bioscience) to determine the activation status of the macrophages (CD206 for M2 and CD86 for M1). Cells were analyzed on a BD LSR Fortessa (BD).
9
Flow Cytometry Analysis of Immune Cell Markers
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Flow cytometry analysis was performed using the following antibodies (BD Bioscience): Anti-mouse CD3-PE/Cy7, Anti-mouse CD11c-PE/Cy7, Anti-mouse CD8-PE/Cy5, Anti-mouse CD44-PE, Anti-mouse CD62L-FITC, Anti-mouse PD-L1-PE, Anti-mouse CD326 PE/Dazzle, Anti-mouse CD4-PE/Dazzle, Anti-mouse CD80-PE, Anti-mouse CD86-PE/Dazzle, and Anti-mouse CD45 FITC. All samples were suspended in FACS buffer and stained with 1 µL antibodies for 30 min at 4°C in darks, then washed twice and resuspended in FACS buffer before analysis. For γ-IFN detection, Human Cytometric Bead Array (CBA) kit and mouse CBA kit (BD Bioscience, USA) were used. The antibody dilution concentration was 1:100. Samples were analyzed with BD Accuri C6 (BD Bioscience, USA) and CytoFLEX (Beckman, USA).
10
Flow Cytometry Analysis of S. aureus Infected BMDCs
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After differentiation, BMDC were plated in 24-well plates at 4 × 105 cells/well and infected with S. aureus strains at the indicated MOI. For surface marker analysis, cells were detached with PBS containing 5 mM EDTA and then incubated in FACS buffer (PBS containing 3% FBS and 0.1% sodium azide). Cells were incubated with purified neutralizing monoclonal antibodies against CD16:CD32 (Fc Block; Biolegend) for 15 min at 4 °C. Next, cells were stained with specific antibodies for 30 min at 4 °C in the dark. The following antibody were used for flow cytometry analysis: anti-Mouse I-A/I-E FITC (BD Biosciences Cat# 553623, RRID:AB_394958), anti-Mouse CD86 PE (BD Biosciences Cat# 553692, RRID:AB_394994), and anti-Mouse CD40 PE (BD Biosciences Cat# 553791, RRID:AB_395055). The stained cells were analyzed by flow cytometry (ACEA NovoCyte Quanteon) with FlowJo 10.4.0 software (TreeStar, Co).
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