Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated percoll density gradient.
A method for the isolation of oligodendroglial cells from young and adult whole rat brains, using a Percoll density gradient is presented. The minced tissue, incubated in a balanced salt solution containing 0.1% trypsin is further dissociated by forcing it through nylon screens to 145 and 74 ?m pore size. The crude suspension is then mixed with an isosmotic Percoll solution and centrifuged for 15 min. An in situ generated density gradient allows the separation of five bands, only one of which (Band C) lying between ?1.050 and ?1.062 contains cellular elements. The isolated cells show the typical morphological characteristics of oligodendroglia. A detailed morphological study of the cells isolated from whole brains of 10-, 30- and 120-day old rats is presented for the first time in the literature and immunocytochemical characterization is carried out using specific (antigalactocerebroside) and non specific (anti-glial fibrillary acidic protein) anti-sera. The method is simple and rapid and isosmotic conditions are maintained throughout, resulting in a better preservation of cell integrity. It represents an improvement over the two previous methods described in the literature and will be useful for studying different developmental events (biochemical and morphological) occurring in oligodendroglial cells at early stages of myelin formation.
Berti Mattera
L N
LN
Departamento de Quimica Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Larocca
J N
JN
de Iraldi
A P
AP
Pasquini
J M
JM
Soto
E F
EF