Magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue.
Pathologically altered functions of astrocytes and microglia play a pivotal role in diseases of the central nervous system (CNS). The complexity of the CNS makes it difficult to determine the function of individual glial cells in vivo. Insight into the role of individual glial cell function lies in their successful isolation and purification to maintain phenotype and realistically mimic in vivo conditions. To facilitate such experiments we have designed a single-step glial cell isolation procedure based on antigen antibody-mediated magnetic cell sorting whereby individual glial cell populations are enriched by positive selection or by depletion from the same mixed glial culture. We removed oligodendroglial contamination from mixed glial culture by antibody-mediated cytolysis, and applied the remaining cells to CD11b MicroBeads in a magnetic field. From the CD11b column we isolated microglia by positive selection and astrocytes by depleting microglia. Microglia isolated by positive selection were >99% pure and free from astrocytes, while astrocytes collected by negative selection were 95-97% pure and completely free from microglia. This modified technique is simple, fast, versatile, convenient and reliable for the isolation of individual glial cell populations from single mixed glial cultures based on cell-specific antigen-antibody interaction. Subsequently, these cultures can be applied to study the function of individual glial cells at the morphological and molecular level during normal and pathological condition.
Marek
Ryan
R
Department of Neurology, School of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Caruso
Michael
M
Rostami
Abdolmohamad
A
Grinspan
Judith B
JB
Das Sarma
Jayasri
J
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000502:physiology*; D001921:Brain / Q000166:cytology*; D039481:CD11b Antigen / Q000378:metabolism; D002452:Cell Count; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005732:Gangliosides / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018189:Immunomagnetic Separation / Q000379:methods*; D045726:Metalloproteases / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000502:physiology*
D039481:CD11b Antigen; D016207:Cytokines; D005732:Gangliosides; D005904:Glial Fibrillary Acidic Protein; C062875:ganglioside A2B5; D045726:Metalloproteases