The following reagents were utilized in the experiments: PE anti-mouse CD11c (Biolegend, Cat. No. 117308, USA), APC anti-mouse CD86 (Biolegend, Cat. No. 105008, USA), FITC anti-mouse CD4 (Biolegend, Cat. No. 100406, USA), APC anti-mouse CD8a (Biolegend, Cat. No. 100712, USA), PE anti-mouse IFN-γ (Biolegend, Cat. No. 505808, USA), and PE anti-mouse TNF-α (Biolegend, Cat. No. 506306, USA).
Apc anti mouse cd86
Manufactured by BioLegend
Sourced in United States
APC anti-mouse CD86 is a fluorochrome-conjugated antibody that binds to the CD86 cell surface molecule expressed on antigen-presenting cells in mice. It can be used for flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pe anti mouse cd11c, by BioLegend (5 mentions)
Fitc anti mouse cd11c, by BioLegend (5 mentions)
Pe anti mouse cd80, by BioLegend (5 mentions)
Fitc anti mouse cd80, by Thermo Fisher Scientific (3 mentions)
Apc anti mouse cd11b, by BioLegend (3 mentions)
Fitc anti mouse f4 80, by BioLegend (3 mentions)
Pe anti mouse ifn γ, by BioLegend (2 mentions)
Fitc anti mouse cd4, by BioLegend (2 mentions)
Fitc anti mouse major histocompatibility complex 2, by Thermo Fisher Scientific (2 mentions)
Fitc anti mouse cd40, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd11c pe, by Thermo Fisher Scientific (2 mentions)
Apc cy7 anti mouse f4 80, by BioLegend (2 mentions)
Apc rat igg2a, by BioLegend (2 mentions)
Pe anti mouse tnf α, by BioLegend (1 mentions)
Apc anti mouse cd8a, by BioLegend (1 mentions)
T0070907, by Selleck Chemicals (1 mentions)
Recombinant rat il 4, by Thermo Fisher Scientific (1 mentions)
Pparγ, by Bioss Antibodies (1 mentions)
P ppar γ, by Bioss Antibodies (1 mentions)
Socs1, by Affinity Biosciences (1 mentions)
19 protocols using apc anti mouse cd86
1
Flow Cytometry of Immune Cell Markers
2
Macrophage Polarization Modulation
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Fluvastatin capsules were purchased from Novartis Pharmaceutical Co., Ltd. (Beijing, China). A Masson staining kit was purchased from Leagene Biotechnology Co., Ltd. (Beijing, China). A hematoxylin-eosin staining kit was purchased from Servicebio Technology Co., Ltd. (Wuhan, China). Cell Counting Kit-8 (CCK-8) was obtained from Fcmacs Biotech Co., Ltd. (Nanjing, China). A BCA protein assay kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Rosiglitazone (RSG, selective PPAR-γ agonist) and T0070907 (selective PPAR-γ antagonist) were obtained from Selleck Chemicals (Houston, USA). LPS was purchased from Sigma Chemical (St. Louis, USA). Recombinant rat IL-4 and INF-γ were purchased from PeproTech Inc. (New Jersey, USA). ELISA kits (IL-1, IL-6, TNF-α, IL4, IL-10, IL-13, and TGF-β1) were obtained from JinYiBai Biological Technology (Nanjing, China). The antibodies (JAK2, STAT6, and p-STAT1) were purchased from Santa Cruz Biotechnology (California, USA). The antibodies (PPAR-γ, STAT1, and p-PPAR-γ) were purchased from Bioss (Beijing, China). The antibodies (SOCS1, SOCS3, p-JAK2, and p-STAT6) were purchased from Affinity Biosciences. APC-anti-mouse CD86 was purchased from BioLegend (San Diego, USA). PE-Cy7-anti-mouse CD206 and Intracellular Fixation & Permeabilization set were purchased from Thermo Fisher Scientific (Massachusetts, USA).
3
Identifying Dendritic Cell Subsets
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Lung single-cell suspensions and BMDC suspensions collected from different groups were stained with PE anti-mouse CD11c (eBioscience, San Diego, CA, USA) and APC-Cy7 anti-mouse F4/80 (Biolegend Inc., San Diego, CA, USA). DCs were marked as CD11c+ and F4/80− cells. This was followed by analysis of the phenotype and the maturation of DCs using FITC anti-mouse CD80, FITC anti-mouse major histocompatibility complex II (MHCII), FITC anti-mouse CD40 (eBioscience, San Diego, CA, USA), and APC anti-mouse CD86 (Biolegend Inc., San Diego, CA, USA) stains. Among them, cDCs were marked as CD11c+MHCII+ double-positive cells. In addition, the cDCs2 (type 2 cDCs) in lung and spleen MNCs were stained as CD11c+CD11b+ double-positive cells using PE anti-mouse CD11c and APC anti-mouse CD11b (Biolegend Inc., San Diego, CA, USA). All the cells, after being washed with PBS, were analyzed using Flow Cytometer (FACSAria™ III, BD Biosciences, USA). The FlowJo software (FlowJo LLC, Ashland, Ore) was used to analyze the data.
4
Optimized Antibody Panel for Immunoblotting and Flow Cytometry
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The following antibodies were used for immunoblotting: Cell Signaling Technology, anti-p38 (Cat#: 8690), anti-p-p38 (Cat#: 4511), anti-p65 (Cat#: 8242), anti-p-p65 (Cat#: 3033), anti-JNK (Cat#: 9252), anti-p-JNK (Cat#: 4668), anti-Erk (Cat#: 4695), anti-p-Erk (Cat#: 4370), anti-PKM2 (Cat#: 4053), anti-STAT3 (Cat#: 12640); Abcam, anti-Pyk2 (Cat#: 228477); Santa Cruz Biotechnology, anti-p-Pyk2 (Cat#: sc-81512); Beyotime Institute of Biotechnology, anti-β-actin (Cat#: AA128), HRP labeled Goat Anti-Rabbit IgG (Cat#: A0208), HRP-labeled Goat Anti-Mouse IgG (Cat#: A0216). The following antibodies purchased from Biolegend were used for flow cytometry: FITC anti-mouse B220 (Cat#: 103206), BV421 anti-mouse CD11c (Cat#: 117330), FITC anti-mouse F4/80 (Cat#: 123108), APC anti-mouse CD86 (Cat#: 105012), PE anti-mouse CD40 (Cat#: 124610), PE anti-mouse GL7 (Cat#: 144607), APC anti-mouse CD95 (Cat#: 152604), APC anti-mouse CXCR5 (Cat#: 145506), PE anti-mouse PD-1 (Cat#: 135206), APC/Cy7 anti-human CD19 (Cat#: 302218), FITC anti-human CD14 (Cat#: 367116), BV421 anti-human CD11c (Cat#: 301628), APC anti-human CD86 (Cat#: 374208), PE anti-human CD40 (Cat#: 334308). All the antibodies for flow cytometry were used at a 1:100 dilution.
5
Mouse Bone Marrow-Derived Dendritic Cell Culture
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BMDCs were obtained by washing the tibiae and femurs of mice with cold PBS and separating cells using a 70-μm cell strainer. The cells were seeded in a 24-well plate (106 cells/well) with conditioned medium (RPMI with 1% penicillin/streptomycin, 10% FBS, glutamine, 1% HEPES, 1% sodium pyruvate, 1% non-essential amino acids, and 50μM β-mercaptoethanol) supplemented with GM-CSF (20 ng/mL, R&D Systems), or GM-CSF VLPs (LD + GM-CSF + 4-1BBL or LD + GM-CSF + OX40L, 5 × 109 particle/mL), and IL-4 (20 ng/mL, R&D Systems). The cells were characterized by flow cytometry labeling surface markers CD11c (FITC anti-mouse CD11c, BioLegend), MHC class II (PE anti-mouse MHC II, BioLegend) and CD86 (APC anti-mouse CD86, BioLegend). The data were analyzed using the ACEA NovoCyte 2000 flow cytometer.
6
Nanoparticle-induced DC Activation
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DCs cultured in vitro were incubated with different nanoparticles (Cro, Cro@aB2MG or CroR@aB2MG) of 0.5 mg/mL under NIR irradiation. After the treatments, cells were collected and subjected to flow cytometry with PE anti-mouse CD80 (104707, BioLegend, China) and APC anti-mouse CD86 (105011, BioLegend, China). The stained cells were quantified with the Flow Jo software.
7
Dendritic Cell Analysis in Murine Spleen and Tumor
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Dendritic cells number was determined in the spleens and tumor tissues by flow cytometry (BD Biosciences, San Jose, CA). The sacrificed mice were soaked in 75% alcohol for 10 minutes and spleens and tumor mass of approximately 5 × 5 mm were collected. Spleens were mechanically dissociated in culture dish and dissociated cells were filtered through a 200-mesh sieve (Solarbio science & technology Co. Ltd. Beijing, China). Splenocytes single-cell suspensions were prepared from each mouse after red blood cells lysis. Tumor tissues were incubated in 1 mL trypsinization buffer at 37°C for 40 minutes after mechanical dissociation. Medium containing serum was added to terminate the tumor tissues digestion and dissociated cells were filtered through a 0.3-μm filter. Subsequently, cells were counted and stained using PE anti-mouse CD11c (1:100, Biolegend, San Diego, CA) to select dendritic cells, FITC anti-mouse CD80 (1:50, Biolegend) or APC anti-mouse CD86 (1:100, Biolegend) to distinguish mature dendritic cells from the immature ones. PE Armenian Hamster IgG (1:100, Biolegend), FITC Armenian Hamster IgG (1:50, Biolegend) and APC Rat IgG2a (1:100, Biolegend) were used as isotype controls.
8
Evaluating Dendritic Cell Maturation
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DC maturation was evaluated by analyzing the expression level of CD86 on the cell surface [19 (link)]. BMDC were seeded in 24-well plates (1 × 105 cells/well), incubated for 24 h, and treated with various nanoparticle formulations at a PIC dose of 5 μg for 24 h. The cells were harvested and stained with FITC-anti-mouse CD11c and APC-anti-mouse CD86 (BioLegend) antibodies for 1 h. For CD11c analysis, the excitation and emission wavelengths used for flow cytometry of FITC were 495 and 519 nm, respectively. For CD86 analysis, the excitation and emission wavelengths used for flow cytometry of APC were 650 and 660 nm, respectively. The expression of CD86 was analyzed using a BD FACSCalibur flow cytometer (BD Bioscience). The expression of surface markers on DC was determined by gating the CD11c+ population.
9
Macrophage Phenotype and JAK/STAT Activation
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Samples were collected, minced, and digested with α-MEM containing 10% FBS in the presence of collagenase I and collagenase II at 37 °C for 1 h. Digested material was filtered with the 70-um strainer and the filtrate was centrifuged at 300 g for 5 min. Next, the collected cells were fixed, permeabilized, and blocked with CD16/32 antibody (Biolegend, CA, USA) on ice for 10 min. To determine JAK/STAT activation in macrophages, the cells were incubated with FITC anti-mouseF4/80 (Biolegend) and primary antibody anti-Phospho-Jak1 (Bioss, Beijing, China), anti-Phospho-JAK3 (Affinity, Jiangsu, China) or anti-Phospho-STAT6 (Abcam, Cambridge, Uk) on ice for 30 min, subsequently incubated with Alexa-Fluor 647 (Abcam) conjugated secondary antibody on ice for additional 30 min. To evaluate macrophages’ phenotype, these cells were stained with FITC anti-mouse F4/80, APC anti-mouse CD86, and PE anti-mouse CD206 antibody (all from Biolegend) on ice for 30 min. Cells that were not treated with any antibody were used as negative controls. The cells were then washed twice to remove excess antibodies, resuspended in 100ul PBS containing 5% FBS, and analyzed by a BD FACSCanto II (New Jersey, USA)
10
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).
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