A procedure for culturing astrocytes from white matter and the application of the siRNA technique for silencing the expression of their specific marker, S100A4.
White matter astrocytes have physiological functions which are distinct from those of astrocytes in gray matter. White matter becomes highly non-permissive to neurite growth after injury, but the role of white matter astrocytes in this process is incompletely understood. Current protocols for making primary astroglial cultures are inadequate for exploring the specific properties of white matter astrocytes in vitro. We describe a procedure for obtaining cultures of white matter astrocytes from the rodent corpus callosum. In this procedure, we take advantage of our previous finding that white, but not gray matter astrocytes express the calcium-binding protein S100A4. S100A4 expressing astrocytes are abundant in the corpus callosum, and we show that cultures, highly enriched in S100A4 expressing white matter astrocytes, can be reproducibly generated from this area. Key factors for successful cultures are (i) meticulous dissection of the corpus callosum from 4-day-old rats, and (ii) Percoll density gradient centrifugation to purify astrocytes. As a means of exploring the possible role of S100A4 in white matter astrocytes, we describe the use of the siRNA technique to eliminate the expression of S100A4 in our in vitro system.
Department of Neuroscience, Neuroanatomy, Biomedical Center, PO Box 587, SE-751 23 Uppsala, Sweden. Elena.Kozlova@anatomi.uu.se
Elena N
EN
Kozlova
Keizo
K
Takenaga
D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000502:physiology; D015415:Biomarkers; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation; D003337:Corpus Callosum / Q000166:cytology; D005260:Female; D020868:Gene Silencing; D008967:Molecular Biology / Q000379:methods*; D011205:Povidone; D011247:Pregnancy; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000235:genetics*; D012822:Silicon Dioxide
D015415:Biomarkers; D034741:RNA, Small Interfering; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C499412:S100a4 protein, rat; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone