Quantitation and distribution analysis of glial fibrillary acidic protein in human glioma cells in culture.
The amount and distribution of glial fibrillary acidic protein (GFAP) were determined by flow cytometry (FCM) and an enzyme-linked immunosorbent assay (ELISA) in five human glioma cell lines stained by the indirect immunofluorescence method using anti-GFAP monoclonal antibody. Standard reference beads containing a known amount of fluorescein were used to calibrate the flow cytometer; however, the intensity of fluorescence from these beads was too weak to allow direct comparison with the fluorescence from the stained cells. Therefore, the flow cytometer was recalibrated using reference beads with a fluorescence intensity similar to that of the glioma cells. By comparing the fluorescence intensities of the two types of reference beads, it was possible to determine the fluorescein content of the stained cells directly from the relative fluorescence intensity (channel number). Glioma cell lines 343 MGA, SF 126, SF 188, U 251, and U 87 had fluorescein concentrations of 72.0 +/- 6.8, 8.1 +/- 0.3, 52.6 +/- 3.1, 86.4 +/- 4.0, and 56.2 +/- 2.9 x 10(5) (mean +/- standard error) Eq Sol Mol (equivalent solution of mole), respectively. The GFAP content of these cell lines, determined by ELISA, was 15.7 +/- 5.2, 0.5 +/- 0.1, 11.1 +/- 2.0, 20.8 +/- 4.6, and 9.5 +/- 2.7 pg GFAP/cell, respectively, and correlated closely with the results of FCM (R = 0.983, p less than 0.0028). A linear regression analysis yielded the following equation: pg GFAP/cell = -2.3376 + 0.2518 x FCM integrated mean channel number (fluorescein concentration: 10(5) Eq Sol Mol).(ABSTRACT TRUNCATED AT 250 WORDS)
Ito
M
M
Department of Neurological Surgery, School of Medicine, University of California, San Francisco.
Nagashima
T
T
Hoshino
T
T
D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D005904:Glial Fibrillary Acidic Protein