Isolation of Mouse Macrophages and Differentiation to M1 and M2 Phenotypes

Jordan Means*, Marieta Gencheva, Hannah Hoblitzell, Nicole Mihalik and Timothy Eubank, Department of Microbiology, Immunology & Cell Biology, West Virginia University, Morgantown, WV 26506

Field (Broad Category): Medical Sciences (Health Sciences) 

Student’s Major: Immunology and Medical Microbiology 

Macrophages found in various tissues arise from different sources. Tissue resident macrophages originate from the yolk sac during development and migrate to tissues to maintain homeostasis. Tissue insults such as acute infections, wound healing, or tumor formation leads to factors being produced to recruit new macrophages from the bone marrow or spleen to resolve such issues. Macrophage function depends on the characteristics of these tissues. Macrophage polarity consists of a pro-inflammatory (“M1”, supporting a Th1 response) or anti-inflammatory (“M2”, supporting a Th2 response) subtype. To generate these cell types, we will flush the bone marrow from mouse femurs, isolate the stem cells, and culture these cells over 5 days with colony-stimulating factor-1 (CSF1) that differentiate these cells into quiescent macrophages (“M0”). After, we will treat some of these M0 macrophages with interferon-gamma (IFN-g) to induce an “M1” response. To generate “M2” macrophages, we will inject thioglycolate into the peritoneal cavity of mice. After 4 days, we will collect and culture these cells with LPS. All cells will be lysed in Trizol to isolate total RNA and synthesize cDNA. We will perform quantitative (q)PCR to determine mRNA expression of genes that represent an “M1” or “M2” response, including IL-10, Nos2, Arg1, Mrc1 and Tgm2. Our ability to collect and differentiate macrophage subtypes allows us to study their activities in vitro. 

Funding: NIH/ National Cancer Institute 

Program/mechanism supporting research/creative efforts: WVU's Research Apprenticeship Program (RAP) & accompanying HONR 297-level course