Intracellular Staining by Flow Cytometry
|Detection of CCL3/MIP‑1 alpha in RAW 264.7 Mouse Cell Line by Flow Cytometry. RAW 264.7 mouse monocyte/macrophage cell line was stained with Rat Anti-Mouse CCL3/MIP‑1 alpha Alexa Fluor® 488‑conjugated Monoclonal Antibody (Catalog # IC450G, filled histogram) or isotype control antibody (Catalog # IC006G, 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.|
The macrophage inflammatory proteins 1 alpha and 1 beta, two closely related but distinct proteins, were originally co-purified from medium conditioned by a LPS-stimulated murine macrophage cell line. Mature mouse CCL3/MIP-1 alpha shares approximately 77% and 70% amino acid identity with human CCL3/MIP-1 alpha and mouse CCL4/MIP-1 beta, respectively. MIP‑1 proteins are expressed primarily in T cells, B cells, and monocytes after antigen or mitogen stimulation. The MIP‑1 proteins are members of the beta (C‑C) subfamily of chemokines.
Both CCL3 and CCL4 are monocyte chemoattractants in vitro. Additionally, the MIP‑1 proteins have been reported to have chemoattractant and adhesive effects on lymphocytes, with CCL3 and CCL4 preferentially attracting CD8+ and CD4+ T cells, respectively. CCL3 has also been shown to attract B cells as well as eosinophils. MIP‑1 proteins have been reported to have multiple effects on hematopoietic precursor cells, and CCL3 has been identified as a stem cell inhibitory factor that can inhibit the proliferation of hematopoietic stem cells in vitro as well as in vivo. In the same assays, CCL4 was reported to be much less active. The functional receptor for CCL3 has been identified as CCR1 and CCR5.