Isolation of murine microglial cells for RNA analysis or flow cytometry.
There is increasing interest in the isolation of adult microglia to study their functions at a morphological and molecular level during normal and neuroinflammatory conditions. Microglia have important roles in brain homeostasis, and in disease states they exert neuroprotective or neurodegenerative functions. To assay expression profiles or functions of microglia, we have developed a method to isolate microglial cells and infiltrating leukocytes from adult mouse brain. This protocol uses a digestion cocktail containing collagenase and dispase, and it involves separation over discontinuous percoll gradients. Isolated cells can be used for RNA analysis, including RNase protection analysis (RPA), quantitative RT-PCR, high-density microarray, proteomic or flow cytometric characterization of cell surface markers or adoptive transfer. Cell isolation can be completed in less than 4 h.
Cardona
Astrid E
AE
Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Huang
DeRen
D
Sasse
Margaret E
ME
Ransohoff
Richard M
RM
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D051379:Mice; D017628:Microglia / Q000502:physiology*; D012313:RNA / Q000302:isolation & purification*
D012313:RNA