IFNβ was purchased from PBL Interferon Source (Piscataway, NJ), and tPA was purchased from Genentech (San Francisco, CA). Evans blue was purchased from Sigma-Aldrich (St. Louis, MO). Triphenyltetrazolium chloride (TTC) and trichloroacetic acid (TCA) were purchased from Alfa Aesar (Tewksbury, MA). Antibodies of Alexa Fluor 488 anti-CD45 (Clone: 30-F11), FITC anti-CD11a (Clone: M17/4), PE/Cy7 anti-Ly6C (Clone: HK1.4), APC anti-CD11b (Clone: M1/70), PE/Cy7 anti-CD68 (Clone: FA-11), PE/Cy7 anti-CD86 (Clone: GL-1), PE/Cy7 anti-CD206 (Clone: C068C2), APC anti-IFNAR1 (Clone: MAR1-5A3), and PE anti-IL-1α (Clone: ALF-161) were purchased from BioLegend (San Diego, CA). APC anti-Arg1 antibody was purchase from R&D Systems. APC anti-IL-1β (Clone: NJTEN3) antibody was purchase from eBioscience (Waltham, MA).
Anti cd86 pe cy7
Manufactured by BioLegend
Sourced in United States
Anti-CD86-PE-Cy7 is a fluorochrome-conjugated monoclonal antibody that binds to the CD86 antigen, also known as B7-2. CD86 is a costimulatory molecule expressed on the surface of antigen-presenting cells, such as dendritic cells, macrophages, and B cells. The PE-Cy7 fluorochrome allows for the detection and analysis of CD86-expressing cells using flow cytometry.
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Lab products found in correlation
Anti cd4 fitc, by BioLegend (4 mentions)
Anti cd11c apc, by BioLegend (4 mentions)
Anti cd206 apc, by BioLegend (3 mentions)
Anti cd8 pe, by BioLegend (3 mentions)
Anti cd80 pe, by BioLegend (3 mentions)
Anti cd3 apc, by BioLegend (3 mentions)
Anti mhcii apc cy7, by BioLegend (3 mentions)
Anti cd11b apc, by BioLegend (2 mentions)
Anti f4 80 fitc, by BioLegend (2 mentions)
Pe anti cd62l, by BioLegend (2 mentions)
Anti cd11b percp cy5, by BioLegend (2 mentions)
Anti cd11c bv650, by BioLegend (2 mentions)
Anti ifn γ pe cy7, by BioLegend (2 mentions)
Anti foxp3 pe cy7, by BioLegend (2 mentions)
Anti cd25 percp cy5, by BioLegend (2 mentions)
Annexin 5 fitc pi, by Dojindo Laboratories (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Dylight 550 nhs, by Thermo Fisher Scientific (2 mentions)
Anti ifn γ pe, by BioLegend (2 mentions)
Anti cd3 fitc, by BioLegend (2 mentions)
18 protocols using anti cd86 pe cy7
1
Immunophenotyping of Mouse Inflammation
2
Phenotyping Dendritic Cells and Macrophages
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The phenotypes of DCs and BMDMs were determined by flow cytometry. For cell surface staining, single-cell suspensions were incubated for 15 min at 4 °C with PE-anti-CD11c (117308, 0.2 μg/mL), FITC-anti-CD80 (104706, 0.2 μg/mL), PE/Cy7-anti-CD86 (105014, 0.2 μg/mL), FITC-anti-MHC-I (114606, 0.2 μg/mL), PerCP/Cy5.5-anti-MHC-II (107626, 0.2 μg/mL), FITC-anti-F4/80 (123108, 0.2 μg/mL), PerCP/Cy5.5-anti-CD11b (101228, 0.3 μg/mL), PE-anti-CD206 (141706, 0.2 μg/mL), APC-anti-CD115 (135509, 0.4 μg/mL), FITC-anti-PDCA-1 (127007, 0.3 μg/mL), PerCP/Cy5.5-anti-CD44 (103032, 0.2 μg/mL), PE-anti-CD62L (104407, 0.2 μg/mL) (all purchased from Biolegend). Samples were washed and then analyzed by FACS versus flow cytometry (BD Biosciences).
For intracellular staining of cytokines, single-cell suspensions from DLN and spleen of the indicated EAE mice were stained with FITC-anti-CD4 (553047, BD Biosciences, 0.2 μg/mL) first, followed by staining with PE-anti-IFN-γ (554412, BD Biosciences, 0.3 μg/mL), PE-anti-IL17A (506904, Biolegend, 0.3 μg/mL) and PE-anti-IL4 (12-7041-82, eBioScience, 0.4 μg/mL) using Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s protocol. Intranuclear staining with PE-anti-Foxp3 (126404, Biolegend, 0.4 μg/mL) was performed using a Fixation/Permeabilization kit (eBioscience) according to the manufacturer’s protocol.
For intracellular staining of cytokines, single-cell suspensions from DLN and spleen of the indicated EAE mice were stained with FITC-anti-CD4 (553047, BD Biosciences, 0.2 μg/mL) first, followed by staining with PE-anti-IFN-γ (554412, BD Biosciences, 0.3 μg/mL), PE-anti-IL17A (506904, Biolegend, 0.3 μg/mL) and PE-anti-IL4 (12-7041-82, eBioScience, 0.4 μg/mL) using Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s protocol. Intranuclear staining with PE-anti-Foxp3 (126404, Biolegend, 0.4 μg/mL) was performed using a Fixation/Permeabilization kit (eBioscience) according to the manufacturer’s protocol.
3
Multi-parameter Flow Cytometry for Splenic Immune Cell Analysis
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Single-cell suspensions from mouse spleens and were stained using the following fluorescence-labeled anti-mouse antibodies: from BD Biosciences, FITC anti-CD38 (BD558813; clone 90; dilution 1:500), BV421 anti-CD95/Fas (BD562633; clone Jo2; dilution 1/500), PE-Cy7 anti-CD86 (BD560582; clone GL1; dilution 1:400), PE anti-CD184/CXCR4 (BD561734; 2B11; dilution 1:250); from BioLegend, APC-Cy7 anti-B220 (103224; clone RA3-6B2; dilution 1:750) and AlexaFluor647 anti-pSer139-H2AX (613407; clone 2F3, dilution 1:200). For internal markers, cells were fixed and permeabilized with the BD Cytofix/Cytoperm fixation/permeabilization solution kit (BD Biosciences). Data were acquired on a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed using the FlowJo software package (BD Biosciences).
4
Identification of Tumor-Infiltrating Immune Cells
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Tumor cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described by Rossowska et al. (30 (link)). Briefly, tumor-derived cells were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher) and then stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 AlexaFluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD86 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE, anti-CD44 PE-Cy7, anti-CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using the FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).
5
Monocyte Immunophenotyping by Flow Cytometry
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Following incubations, the plate was incubated in 4°C for 30 min and rubbed gently by pipette. Then, suspended monocytes were collected and transferred to FACS tubes. Then, cells were centrifuged and washed with 1 ml FACS buffer. Pelleted cells were surface stained with anti-CD14-BV421, anti-CD16-BV510, anti-CD86-PE-Cy7, anti-HLA-DR-PE, anti-CD206-APC, and anti-CD163-BV605 (BioLegend) for 30 min at 4°C. After washing with FACS buffer, cells were fixed by 4% paraformaldehyde (Sigma, UK) for 10 min at 4°C and then washed by FACS buffer. Finally, cells were resuspended in 250 μl FACS buffer and kept in 4°C until analysis. Data was acquired using an LSRII flow cytometer (BD Biosciences) configured with three lasers and 10 detectors and FACSDiva acquisition software (BD Biosciences). Compensation was performed using tubes of CompBeads (BD Biosciences) individually stained with each fluorophore and compensation matrices were calculated with Flowjo version 10 (TreeStar Inc., Ashland, OR, USA).
6
Glycosylated-PEG Polymer for Immunomodulation
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Galactose-polyethylene glycol polymer chain (glycosylated-PEG, 35kDa) was purchased from Tianjin Jenkem Technology Co. Ltd (Tianjin, China). Annexin V-FITC/PI and CCK8 kit were purchased from Dojindo Laboratories. Anti-CD11c-APC, anti-CD4-FITC, anti-CD86-PE-Cy7, anti-CD3-APC, anti-Foxp3-PE-Cy7, anti-IFN-γ-PE-Cy7, anti-CD80-PE, anti-CD8-PE, anti-3-FITC, anti-NK1.1-APC, and anti-CD25-Percp-Cy5.5 were purchased from BioLegend, Inc (San Diego, CA, USA). LDH kits were purchased from Abcam (Cambridge, UK).
7
Pulmonary Macrophage Phenotyping
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Lungs obtained at 7 dpi were injected with saline solution to obtain cells. Red blood cells were lysed, and live cells were counted using trypan blue exclusion (Countess II FL ThermoFisher). The cells were stained for expression of surface markers using the following antibodies: anti-F4/80-Pacific blue or APC, anti-CD86-PE/Cy7, anti-CD206 (MMR)-PerCP or FITC and anti-IL4Rα-PE (all from Biolegend; CA, USA). The samples were incubated for 30 min at 4 °C in FACS sheet buffer (BD®). The samples were acquired using either a FACSAria Fusion (BD®) or Attune NxT (ThermoFisher) and analyzed using FlowJo software (version 10.0.7).
8
Multimodal Nanoplatform for Cancer Theranostics
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Silicon phthalocyanine dichloride (SiPCCl2), 3‐aminopropyltrimethoxysilane (APTES), Hoechst 33342, tetraethylorthosilicate (TEOS), and 2, 7‐dichlorodihydroflfluorescein diacetate (DCFH‐DA) were obtained from Sigma‐Aldrich. Hexadecyl trimethylammonium chloride (CTAC), triethanolamine (TEA), and FeCl3 were obtained from Sinopharm Chemical Reagent Co. Ltd. Dylight550‐NHS was purchased from Thermo Fisher Scientific (Waltham, USA). ICG‐NHS was obtained from Xi'an Rixi Biological Technology Co. Ltd (Xi'an, China). Calcein‐AM/propidium iodide (PI), and 1, 3‐diphenylisobenzofuran (DPBF) were obtained from KeyGen BioTech (Shanghai, China). Annexin V‐FITC/PI and CCK8 were obtained from Dojindo Laboratories. Annexin V‐APC/PI was obtained from Jiangsu KeyGEN BioTECH Corp., Ltd (Jiangsu, China) Neutrophil (mouse) isolation Kit was purchased from Cayman Chemical (Michigan, USA). Anti‐CD11c‐APC, anti‐CD137‐APC, anti‐CD11b‐APC, anti‐CD86‐PE‐Cy7, antiCD3‐APC, anti‐CD80‐PE, anti‐CD8‐PE, anti‐Ly‐6G/Ly‐6C‐PE, CD54, CD95, anti‐CD8 antibody, and anti‐CD69‐FITC was purchased from BioLegend, Inc (San Diego, CA, USA). ELISA kits from Neobioscience Technology (Shenzhen, China) were obtained to detect the IFN‐γ, TNF‐α, IL‐12, and IL‐2. H22 mouse liver cancer cell‐specific neoantigen (sequence: HTDAHAQAFAALFDSMH) was obtained from GenScript USA Inc.
9
Multiparametric Flow Cytometry Analysis
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All cells were labeled with live/dead dye near infra red (Life Technologies) for dead cell exclusion and treated with Fc Block (Miltenyi, San Diego, CA, USA) prior to staining with fluorescently labeled antibodies. Anti-human antibodies used for DC staining were anti-CD83 PE (Biolegend, San Diego, CA, USA), anti-CD86-PE-Cy7 (Biolegend), anti-HLA-DR V450 (BD), and anti-CD11c APC (BD). Antibodies used in the T cell characterization were anti-LAG-3 FITC (Novus, Littleton, CO, USA), anti-PD-1 PerCP-Cy5.5, anti-CD3 Alexa700, anti-CD4 Brilliant Violet 650, anti-CD8 Brilliant Violet 570, anti-IFN-γ PE, anti-IL-5, anti-CD62L PE, and anti-CD45RA Alexa700 (all, Biolegend), anti-CD25 PE, and anti-IFN-γ PE-Cy7, anti-IL-4 PE-Cy7 (all BD), and anti-TIM-3 APC (R&D Systems). Surface staining and intracellular staining was performed using Cytofix/Cytoperm™ Plus kit (BD) according to the manufacturer’s instructions. Data acquisition was performed using the LSRFortessa flow cytometer (BD). Data analysis was performed with FlowJo version 9 (Tree Star, Inc., Ashland, OR, USA).
10
Neutrophil-Derived Extracellular Vesicles Modulate Macrophage Responses
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Extracellular vesicles (30 µg total protein/ml), which were produced by non-stimulated neutrophils (EV-NS), or by neutrophils stimulated with PMA (EV-PMA), fMLF (EV-fMLF), or Mtb (EV-TB) for 30 min, were used to stimulate macrophages (2 × 105) for 24 h. As controls, the macrophages were left with medium alone (NS) or were infected with Mtb (2 × 106). After this incubation, the supernatants were collected, centrifuged at 400 × g at 4°C and stored at −20°C until analysis. IL-1β, IL-6, IL-10, and TNF-α were measured with a cytometric bead array (BD), and TGF-β was measured with an ELISA Kit (BioLegend), according to the manufacturer’s protocol. In the same experiments, macrophages (detached from the culture plate with cold PBS) were washed and centrifuged at 400 × g at 4°C, and stained with anti-CD14/APC, Lin1 (anti-CD3, CD14, CD16, CD19, CD20 and CD56)/FITC, anti-HLA-DR/FITC, anti-CD1a/PE, anti-CD11c/PB, anti-CD80/PE-Cy5, anti-CD86/PE-Cy7, and the corresponding isotype controls (BioLegend), for 15 min at 4°C. Cells were then washed with 1% BSA in PBS and analyzed by flow cytometry.
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