A simple method to increase the proportion of bone marrow-derived macrophages positive for M-CSFR using the reducing agent dithiothreitol (DTT)

Abstract

Only a few bone marrow-derived macrophages (BM-MΦ) are positive for macrophage colony-stimulating factor receptor (M-CSFR). Thus, a method is needed to increase the proportion of BM-MΦ that are positive for M-CSFR to facilitate the investigation of the effects of M-CSFR downregulation on various diseases. We used mouse primary BM-MΦ to evaluate the expression of M-CSFR on the cytoplasmic membrane using flow cytometry. Treatment with a reducing agent, dithiothreitol (DTT), increased the proportion of BM-MΦ that were positive for M-CSFR, and this increase was time dependent. We next determined whether DTT-treated BM-MΦ can lead to the downregulation of M-CSFR. Treatment with lipopolysaccharide (LPS) for 24 h. decreased the proportion of DTT-treated BM-MΦ that were positive for M-CSFR. These results suggest that DTT treatment increases the proportion of BM-MΦ that are positive for M-CSFR and that the upregulation of M-CSFR on BM-MΦ can be abrogated by treatment with LPS. Here, we propose a simple method to increase the number of M-CSFR-positive BM-MΦ using the reducing agent DTT, which could be useful in investigations of the relationship between the downregulation of M-CSFR and some diseases. • The proportion of BM-MΦ that expresses M-CSFR on the membrane increases by approximately twice following DTT treatment. Method name: Method to increase the proportion of macrophages positive for M-CSFR, Keywords: Bone marrow-derived macrophages, Macrophage colony-stimulating factor receptor (M-CSFR), Dithiothreitol (DTT), Lipopolysaccharide (LPS), Atherosclerosis