QUANTIFICATION OF NEUROTOXICITY AND IDENTIFICATION OF CELLULAR SUBSETS IN A 3-DIMENSIONAL BRAIN MODEL

Imaging of cells in a large intact three-dimensional tissue remains di fficult. Quantification and identification of cell, damage in a mixed culture system has been limited by the inability of fluorescent probes to discriminate types of cellular death and penetrate tissue more tha t 100 mu m thick. We have investigated several probes in combination w ith neural cell-specific antibodies to quantify cell damage in the pre sence of several toxins. Acridine orange and ethidium bromide were exc ellent for determination of cell viability, death by necrosis, or apop tosis in thick brain tissue aggregates. Calcein and ethidium homodimer were effective on live/dead stains, and the Syto dyes 11 and 13 worke d well for quantification of all cells In. the brain aggregate model. By using these combinations of dyes in conjunction with confocal micro scopy, we mere able to quantify neural cell damage without disrupting the three-dimensional environment. (C) 1998 Wiley-Liss, Inc.