Application of flow cytometry to analyses of cultured human glioma and fetal brain cells.
Methods are described to study cell surface and cytoplasmic antigens of cultured human glioma, fetal brain cells and fibroblasts using flow cytometry. This required harvesting the cultured cells with Versene or mild trypsin treatment and fixation in 4% paraformaldehyde prior to staining for glial fibrillary acidic protein (GFAP) and fibronectin using indirect immunofluorescence. At passage 10, 38% of fetal brain cells [CHII] were GFAP-positive but at passage 14 only 3.5% expressed GFAP. Two glioblastomas and an anaplastic astrocytoma had 38.8%, 6.7% and 81.3% GFAP-positive cells, respectively. Of the 10(4) cells studied, 91.6%, 79.1% and 40.8% were fibronectin-positive for glioblastoma multiforme [12-18], oligodendroglioma [12-10] and fetal brain [CHII] cells, respectively. Two fibroblast lines had 33.5% and 43.1% of the cells expressing fibronectin. The validity of these results was confirmed by staining for GFAP and fibronectin using peroxidase-antiperoxidase and immunofluorescence microscopy. Using low angle forward light scatter to estimate cell size and gating techniques it was found that GFAP-positive CHII and anaplastic astrocytoma cells were generally larger than GFAP-negative cells of the same type. No correlation between cell size and fibronectin expression was found for glioblastoma [12-18] cells. These results demonstrate the validity of the described methods and illustrate some specific applications and the potential value of flow cytometry to neurooncology.
D001932:Brain Neoplasms / Q000032:analysis / Q000473:pathology; D002460:Cell Line; D005333:Fetus; D005353:Fibronectins / Q000032:analysis; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D005910:Glioma / Q000032:analysis / Q000473:pathology*; D006801:Humans; D007124:Immunoenzyme Techniques
D005353:Fibronectins; D005904:Glial Fibrillary Acidic Protein