Monocytes were seeded in a 96-well plate at 3x105 cells per well and differentiated to MΦ1, MΦ2 and DCs as described above. After 7 days, the cells were challenged with 500ng/ml LPS (Sigma) and surface markers were determined. Therefore, the cells were detached from the plate using 5mM EDTA (20 minutes at 37°C). The cells were spun down at 300xg, the EDTA was discarded and the cells were incubated with a mix of anti-CD14-APC-Cy7, anti-CD209-PerCP-Cy5.5, anti-CD163-PE and anti-CD80-PE-Cy7 (5μl of each antibody, all antibodies from BD). The antibodies were incubated for 20 minutes at 4°C. After incubation, 200μl PBS was added and the cells were spun down at 300xg. The supernatant was discarded, 200μl PBS was added once more and the cells were collected by centrifugation. Readout was done on a BD FACS CANTO II and subsequent analysis were done using BD DIVA software. Plots were made in Sigmaplot (Systat Software).
Anti cd80 pe cy7
Manufactured by BD
Sourced in Netherlands
Anti-CD80-PE-Cy7 is a fluorescently-labeled monoclonal antibody that binds to the CD80 cell surface protein. CD80 is a co-stimulatory molecule expressed on antigen-presenting cells. This product is intended for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Diva software, by BD (1 mentions)
Anti cd163 pe, by BD (1 mentions)
Anti cd14 apc cy7, by BD (1 mentions)
Sigmaplot, by Grafiti LLC (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti cd86 apc, by BD (1 mentions)
Pe cy7, by BD (1 mentions)
Anti mhcii apc, by Thermo Fisher Scientific (1 mentions)
Anti cd40 pe, by Beckman Coulter (1 mentions)
Anti pd l1 pe, by BD (1 mentions)
Bdca2 pe, by Miltenyi Biotec (1 mentions)
Bdca 4 pe, by Miltenyi Biotec (1 mentions)
Cd123 apc, by Miltenyi Biotec (1 mentions)
Migg1 apc, by BD (1 mentions)
Anti cd83 pe, by Beckman Coulter (1 mentions)
Migg1 pe, by BD (1 mentions)
Anti cd80 pe, by BD (1 mentions)
Clone g46 2, by BD (1 mentions)
Anti cd86 pe cy7, by BD (1 mentions)
3 protocols using anti cd80 pe cy7
1
Differentiation and Characterization of Monocyte-Derived Immune Cells
2
Phenotypic Analysis of DC Subsets
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Purity of pDCs and mDCs after isolation and the phenotype of the pDC populations were determined by flow cytometry. The following primary monoclonal antibodies (mAbs) and the appropriate isotype controls were used: anti-BDCA1-FITC, BDCA2-PE, BDCA4-PE and CD123-APC (all Miltenyi Biotec); mIgG1-PE, mIgG1-APC, anti-CD11c-FITC or -APC, anti-HLA-ABC-PE (W6/32), anti-CD80-PE, or -APC, or -PeCy7, anti-CD86-PE, or -APC (all BD Bioscience Pharmingen, San Diego, CA, USA) anti-PD-L1-APC, anti-PD-L2-PE; anti-CD40-PE, anti-CD83-PE (Beckman Coulter, Mijdrecht, the Netherlands); anti-MHC-II-APC (eBioscience).
The phenotype of the DC populations after treatment with vemurafenib, dabrafenib, trametinib or a combination was determined by staining with the following antibodies and appropriate isotype controls: anti-CD80-PE-Cy7, anti-CD86 APC, anti-PD-L1-PE, anti-HLA-ABC-V450, anti HLA-DR-BV510, mIgG1-PE-Cy7, mIgG1-PE (all BD biosciences) and mIgG1-APC (eBioscience).
The phenotype of the DC populations after treatment with vemurafenib, dabrafenib, trametinib or a combination was determined by staining with the following antibodies and appropriate isotype controls: anti-CD80-PE-Cy7, anti-CD86 APC, anti-PD-L1-PE, anti-HLA-ABC-V450, anti HLA-DR-BV510, mIgG1-PE-Cy7, mIgG1-PE (all BD biosciences) and mIgG1-APC (eBioscience).
3
Phenotypic Analysis of DC Maturation
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Phenotypical assessment of DC maturation after 48 h of co-culture with tumor cells was performed by flow cytometry. Briefly, cells were washed in PBA, incubated with Fc-receptor blocking buffer (2% HS in PBS, 15 min at 4°C) and subsequently stained with primary antibodies in PBA (30 min at 4°C). Monoclonal directly labeled anti-human antibodies used were (Table S1): anti-CD11c-APC (Miltenyi biotec, clone MJ4-27G12, catalog# 130-092-412), anti-CD123-APC (Miltenyi biotec, clone AC145, catalog# 130-090-901), anti-CD80-PECy7 (BD PharMingen, clone L307.4, catalog# 561135), anti-CD86-PECy7 (BD PharMingen, clone 2331, catalog# 561128), anti-HLA-ABC-PE (BD PharMingen, clone G46-2.6, catalog# 555553), and anti-HLA-DR-PE (BD PharMingen, clone G46-6, catalog# 555812). Appropriate isotype controls were included. Geometric mean fluorescence intensity (GeoMFI) of maturation markers was assessed on CD11c+ (for CD1c+ and CD16+ DCs) or CD123+ (for pDCs) populations. As a positive control, DCs were stimulated with poly I:C (CD1c+ and CD16+ DCs, 2 µg/mL, Enzo Life Science, catalog# ALX-746-021-M005) or R848 (pDCs, 4 µg/mL, Enzo Life Science, catalog# ALX-420-038-M025). To improve in vitro pDC viability, IL-3 (10 ng/mL, Cellgenix, catalog# 1002-050) was added to culture medium.
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