Intracellular Staining by Flow Cytometry
|Detection of CXCL9/MIG in rhIFN gamma -treated THP‑1 Human Cell Line by Flow Cytometry. THP‑1 human acute monocytic leukemia cell line was treated for 24 hr with 100 U/mL rhIFN gamma then stained with Mouse Anti-Human CXCL9/MIG PE‑conjugated Monoclonal Antibody (Catalog # IC392P, filled histogram) or isotype control antibody (Catalog # IC002P, open histogram). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.|
CXCL9, a member of the alpha subfamily of chemokines that lack the ELR domain, was initially identified as a lymphokine-activated gene in mouse macrophages. Human CXCL9 was subsequently cloned using mouse MIG cDNA as a probe. The CXCL9 gene is induced in macrophages and in primary glial cells of the central nervous system specifically in response to IFN-gamma. CXCL9 has been shown to be a chemoattractant for activated T-lymphocytes and TIL but not for neutrophils or monocytes. The human CXCL9 cDNA encodes a 125 amino acid (aa) residue precursor protein with a 22 aa residue signal peptide that is cleaved to yield a 103 aa residue mature protein. CXCL9 has an extended carboxy-terminus containing greater than 50% basic aa residues and is larger than most other chemokines. The carboxy-terminal residues of CXCL9 are prone to proteolytic cleavage resulting in size heterogeneity of natural and recombinant CXCL9. CXCL9 with large carboxy-terminal deletions have been shown to have diminished activity in the calcium flux assay. CXCL9 is a homodimer and will heterodimerize with CXCL12. It is reported to bind to CXCR3, CXCR6 and CCR3. Over amino acids (aa) 23-125, human and mouse CXCl9 share 67% aa sequence identity.
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