DC2.4 cells were seeded overnight in a 48-well plate at 5 × 104 cells per well in complete RPMI media. Cells were treated for 16 hours with either 20 nmol of freshly prepared mPEG2000−Q11OVA nanofiber solutions or with tablets containing the same quantity of nanofibers. Formulations were unadjuvanted or contained CTB at 50 μg/mL. Cells were stained with I-A/I-B:FITC (BioLegend, cat #107606), CD80:PE (BioLegend, cat #104708), CD86:APC/Cy7 (BioLegend, cat# 105030), and DAPI. Flow cytometry was performed on a FACS Canto cytometer and data was analyzed using FlowJo software.
Cd86 apc cy7
Manufactured by BioLegend
CD86-APC-Cy7 is a fluorescently-labeled antibody that binds to the CD86 surface protein. CD86 is a costimulatory molecule expressed on antigen-presenting cells that plays a key role in T cell activation and differentiation. The APC-Cy7 fluorescent label allows for detection and analysis of CD86-expressing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd80 pe, by BioLegend (3 mentions)
Cd14 pe cy7, by BioLegend (2 mentions)
Cd40 apc, by BioLegend (2 mentions)
Mhcii fitc, by BioLegend (2 mentions)
Cd11a pe, by BioLegend (2 mentions)
Pd l1 bv421, by BioLegend (2 mentions)
Tlr2 apc, by BioLegend (2 mentions)
Mrc1 pe, by BioLegend (2 mentions)
Siglec1 fitc, by BioLegend (2 mentions)
Lsr 2, by BD (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Cd11c apc, by BD (1 mentions)
Cd11b percp, by BioLegend (1 mentions)
Cd115 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd206 pe cy7, by BioLegend (1 mentions)
Cd36 pe, by BioLegend (1 mentions)
Facs canto 2 cytometre, by BD (1 mentions)
Cd69 pe, by BioLegend (1 mentions)
Quantikine elisa, by R&D Systems (1 mentions)
6 protocols using cd86 apc cy7
1
Nanofiber-based Antigen Delivery Analysis
2
Immunophenotyping of Immune Cells
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For all experiments, cells were lifted by gentle scrapping, washed with PBS, and stained with MHCII-FITC (BioLegend, Cat no. 107606), CD40-APC (BioLegend, Cat no. 124612), PDL1-BV421 (BioLegend, Cat no. 124315), CD80-PE (BioLegend, Cat no. 104708), and CD86-APC-Cy7 (BioLegend, Cat no. 104708) TLR2-APC (Biolegend, Cat no. 153006) MRC1-PE (Biolegend, Cat no. 141706) Siglec1-FITC (Biolegend, Cat no. 142406) CD14-PE-Cy7 (Biolegend, Cat no. 123316) CD11a-PE (Biolegend, Cat no. 153103) (all diluted 1:400 in PBS). Cells were then washed 3 times in PBS and fixed with 1% formaldehyde (J.T. Baker, Cat no. JTB-2106-01) in PBS. Flow cytometry was performed on a BD LSR II or an Attune CytPix at the MSU Flow Cytometry Core, and data were analyzed using FlowJo (Version 10.8.1).
3
Immunomodulatory Effects of NSAIDs
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Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St Louis, MO, USA), and used at a concentration of 0.5 μg/ml. CpG-ODN 1668 (‘5-TCCATGACGTTCCTGATGCT-3’ ) with total phosphorothioate-modified backbone was purchased from Sigma Genosys (Haverhill, UK), and used at a concentration of 1 μg/ml. R848 was purchased from Enzo Life Sciences, and used at a concentration of 4 μg/ml. The NSAIDs diclofenac sodium salt (PHR1144-1G), celecoxib (PHR1683-1G), and ibuprofen (PHR-1004-1G) were purchased from Sigma-Adrich. For every experiment, a fresh dilution in medium was made. Celecoxib was first diluted in DMSO and thereafter further diluted in medium (DMSO concentration <0.03%). For flow cytometry experiments the following antibodies (clone name in brackets, followed by supplier, and dilution) conjugated to various fluorophores were used: MHCII-BV510 (M5/114.15.2, Antibodychain, 1:500), CD80-A488 (16-10A1, Antibodychain, 1:1000), CD11b-PerCP (M1/70, Biolegend, 1:600), CD115-PeCy7 (AFS98, eBioscience, 1:200), CD11c-APC (HL3, BD, 1:400), and CD86-APCCy7 (GL-1, Biolegend, 1:800). Viability dye used was eFluor™ 450 (ThermoFisher, 1:4000).
4
Phenotyping Murine Bone Marrow-Derived Macrophages
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BMDMs were obtained from control and ketamine-treated mice as previously described [35] . Briefly, cells were cultured in RPMI 1640 supplemented with 13% supernatant of the mouse L929 cell line (conditioned medium) for 7 days. Then, cells were stimulated with LPS (100 ng/mL) or maintained in media for 24 h. Cells were harvested and stained with CD86-APC/Cy7 (BioLegend Cat # 105,029, RRID: AB_2074993), CD206-PE/Cy7 (BioLegend Cat # 141719, RRID: AB_2562247), and CD36-PE (Biolegend Cat # 102605, RRID: AB_389348) antibodies before analysis by flow cytometry with the FACS Canto II cytometre (Becton Dickinson).
5
Multiparametric Flow Cytometry Analysis
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For all experiments, the cells were lifted by gentle scraping, washed with PBS, and stained with MHC-II-FITC (BioLegend, catalog no. 107606), CD40-APC (BioLegend, catalog no. 124612), PDL1-BV421 (BioLegend, catalog no. 124315), CD80-PE (BioLegend, catalog no. 104708), CD86-APC-Cy7 (BioLegend, catalog no. 104708), TLR2-APC (BioLegend, catalog no. 153006), MRC1-PE (BioLegend, catalog no. 141706), Siglec1-FITC (BioLegend, catalog no. 142406), CD14-PE-Cy7 (BioLegend, catalog no. 123316), CD11a-PE (BioLegend, catalog no. 153103), and CD69-PE (BioLegend, catalog no. 104508) (all diluted 1:400 in PBS). The cells were then washed three times in PBS and fixed with 1% formaldehyde (J. T. Baker, catalog no. JTB-2106-01) in PBS. Flow cytometry was performed on a BD LSR II or an Attune CytPix at the Michigan State University Flow Cytometry Core, and the data were analyzed using FlowJo (version 10.8.1).
6
Murine Dendritic Cell Phenotyping
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The commercial sources of cell culture reagents were as follows: RPMI 1640 culture media (BioloT), serum-free and phenol-red-free Opti-MEM (Thermo Fisher Scientific), fetal calf serum (FCS; HyClone), 2-mercaptoethanol (Sigma), L-glutamine (BioloT), bovine insulin (Pan-Eco), gentamicin (KRKA), benzylpenicillin (Sintez), HEPES buffer (Sigma), propidium iodide (PI) (Sigma), and 20% (v/v) Tween (Sigma). Monoclonal antibodies for DC phenotyping were purchased from BioLegend (CD11c-FITC, H2b-PE, H2k-PE, CD86-APC-Cy7, CD80-Brilliant Violet 450, CD4-PerCP, CD25-APC, IL-10-PE, and FoxP3-PE). Recombinant murine cytokines (IL-4, GM-CSF) were procured from R&D Systems. The mouse IL-10 quantitation immunoassay (Quantikine ELISA) was procured from R&D Systems. The colorimetric PreMix WST-1 assay to assess cell proliferation was purchased from Takara (Japan). Culture flasks and plates were from TPP (Switzerland), Petri dishes were from Nunclon, and monensin and brefeldin A were from Sigma.
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