Subpopulations of WBC were analyzed using a combination of side scatter (SSC) characteristics and CD45-APC/Cy7 (HI30) (purchased from BioLegend, San Diego, CA). Murine peripheral blood cells and bone marrow cells were stained with CD11b conjugated with FITC (clone: M1/70), CD45-APC/Cy7 (clone: 30-F11) and Ly6G-PeCy7(clone: 1A8) (all purchased from BioLegend, San Diego, CA). Viable and non-viable cells were distinguished using DAPI.
Cd45 apc cy7 hi30
Manufactured by BioLegend
CD45-APC/Cy7 (HI30) is a fluorochrome-conjugated antibody that binds to the CD45 antigen. CD45 is a transmembrane glycoprotein expressed on the surface of all leukocytes. The antibody is conjugated with APC/Cy7, a fluorescent dye combination that can be detected using flow cytometry or other relevant analytical techniques.
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2 protocols using cd45 apc cy7 hi30
1
Murine Peripheral Blood and Bone Marrow Analysis
2
Flow Cytometric Analysis of Immune Cells
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After red blood cell lysis, WBCs (106/mL) were treated with Human TruStain FcX (BioLegend, San Diego, CA) to prevent nonspecific binding, followed by incubation with relevant antibodies for 25 minutes at 4°C. Subpopulations of WBCs were analyzed using the following antibodies: fluorescein isothiocyanate–conjugated CD3 (UCHT1), PE (Phycoerythrin)‐conjugated CD73 (AD2), CD19‐PE/Cy7 (HIB19), CD39‐APC (Allophycocyanin) (A1), and CD45‐APC/Cy7 (HI30) (all from BioLegend).
For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA). PE‐conjugated anti‐human TNF‐α (MAb11) and IgG1‐PE (MOPC‐21) isotype‐matched control antibodies (BioLegend) were used to determine intracellular level of TNF‐α protein. Data acquisition was performed on a MacsQuant Analyzer 10 (Miltenyi Biotec, Inc), and the data were analyzed using WinList 5.0 software. Viable and nonviable cells were distinguished using 4’,6‐diamidino‐2‐phenylindole or LIVE/DEAD Fixable Blue Stain kit (Life Technologies, Carlsbad, CA).
For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA). PE‐conjugated anti‐human TNF‐α (MAb11) and IgG1‐PE (MOPC‐21) isotype‐matched control antibodies (BioLegend) were used to determine intracellular level of TNF‐α protein. Data acquisition was performed on a MacsQuant Analyzer 10 (Miltenyi Biotec, Inc), and the data were analyzed using WinList 5.0 software. Viable and nonviable cells were distinguished using 4’,6‐diamidino‐2‐phenylindole or LIVE/DEAD Fixable Blue Stain kit (Life Technologies, Carlsbad, CA).
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