Leukocytes (~1×106 cells/tube) were stained with DAPI (cell viability), anti-CD45-Pacific Blue, anti-F4/80-FITC, anti-CD11b-PercP Cy5.5, anti-Ly6G-APC, anti-CXCR2-PE (all R&D Systems), anti-CD62L-FITC, anti-GRK2-FITC, anti-Cytokeratin (C-11)-FITC (Abcam), anti-IL-33-PE (R&D Systems) and isotype controls (BD Biosciences), following the manufacturer’s protocols. IL-33 production by CD45− Cytokeratin+ epT cells was determined after stimulation with PMA (50 ng/ml) and ionomycin (1 μg/ml) in the presence of GolgiStop™ and permeabilization of the cells (Cytofix/Cytoperm, BD Biosciences). Cells were acquired using a Beckman Coulter CyAn™ ADP (Beckman Coulter, USA). Gating strategy (Supplementary Figure S5 online ) and analysis were performed using the FlowJo® software (treeStar Software, USA).
Anti ly6g apc
Manufactured by R&D Systems
Anti-Ly6G-APC is a fluorescently-labeled antibody that binds to the Ly6G antigen, which is expressed on the surface of mature neutrophils. This product can be used for the identification and characterization of neutrophils in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Isotype control, by BD (2 mentions)
Anti cd45 pacific blue, by R&D Systems (2 mentions)
Anti f4 80 fitc, by R&D Systems (2 mentions)
Anti cd11b percp cy5, by R&D Systems (2 mentions)
Anti cxcr2 pe, by R&D Systems (2 mentions)
Anti cd62l fitc, by Abcam (2 mentions)
Anti grk2 fitc, by Abcam (2 mentions)
Anti cytokeratin c 11 fitc, by Abcam (2 mentions)
Anti il 33 pe, by R&D Systems (2 mentions)
Cyan adp, by Beckman Coulter (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Anti cd11b pe cy7, by BioLegend (1 mentions)
Anti cd45 percpcy5, by R&D Systems (1 mentions)
Cd31 pe, by R&D Systems (1 mentions)
Anti cd206 apc, by BioLegend (1 mentions)
Anti cd45 percp cy5, by BioLegend (1 mentions)
Anti f4 80 pe, by Bio-Rad (1 mentions)
Anti ly6c apc, by BioLegend (1 mentions)
Zombie dye, by BioLegend (1 mentions)
Facslyric, by BD (1 mentions)
3 protocols using anti ly6g apc
1
IL-33 Production in Epidermal T Cells
2
Multiparametric Flow Cytometry of Myeloid Cells
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Cells were incubated with antibody mixture including anti-F4/80-PE (BioRad), anti-Ly6G-FITC, anti-CD11b-PECy7, anti-CD45-PerCPCy5.5 and anti-Ly6C-APC or, anti-CD301-APC or anti-CD206 APC (Biolegend, San Diego, CA, USA) for 15 min and subsequently, 0.4 µl zombie dye (Biolegend) was added for 5 min. For detection of S100A9 protein intracellular staining was performed using the True-Nuclear™ Transcription Factor Buffer Set. Cells were first stained with antibody mixture including anti-CD45-PerCPCy5.5, anti-CD11b-PECy7, anti-Ly6G-APC, anti-F4/80-PE or anti-CD45-PerCPCy5.5, CD31-PE, CD90-PE-Cy7 for 15 min, washed and fixed for 45 min before intracellular staining with goat anti-S100A9 antibody (R&D) for 30 min, washing and addition of secondary antibody (anti-goat Alex 488, Life Technology) for further 30 min.
Flow cytometry was performed with BD FACSCantoTMII or BD FACSLyric™ and data analysed with BD FACSDivaTM Software or BD FACSuite™ Application (BD, Heidelberg, Germany). Using forward and sideward scatter single cells were gated. Living singlets were chosen from zombie-negative cells as described 31 (link).
Flow cytometry was performed with BD FACSCantoTMII or BD FACSLyric™ and data analysed with BD FACSDivaTM Software or BD FACSuite™ Application (BD, Heidelberg, Germany). Using forward and sideward scatter single cells were gated. Living singlets were chosen from zombie-negative cells as described 31 (link).
3
IL-33 Production in Epidermal T Cells
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Leukocytes (~1×106 cells/tube) were stained with DAPI (cell viability), anti-CD45-Pacific Blue, anti-F4/80-FITC, anti-CD11b-PercP Cy5.5, anti-Ly6G-APC, anti-CXCR2-PE (all R&D Systems), anti-CD62L-FITC, anti-GRK2-FITC, anti-Cytokeratin (C-11)-FITC (Abcam), anti-IL-33-PE (R&D Systems) and isotype controls (BD Biosciences), following the manufacturer’s protocols. IL-33 production by CD45− Cytokeratin+ epT cells was determined after stimulation with PMA (50 ng/ml) and ionomycin (1 μg/ml) in the presence of GolgiStop™ and permeabilization of the cells (Cytofix/Cytoperm, BD Biosciences). Cells were acquired using a Beckman Coulter CyAn™ ADP (Beckman Coulter, USA). Gating strategy (Supplementary Figure S5 online ) and analysis were performed using the FlowJo® software (treeStar Software, USA).
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