Quantitative analysis of turbellarian cell suspensions by fluorescent staining with acridine orange, and video microscopy

A combination of methods has been developed for analyzing cell suspensions from turbellarians with respect to cytochemical and morphometric parameters and with special emphasis on the characterization of neoblasts. Tissues were disintegrated by mechanical and enzymatic means. Neoblasts were separated and/or fractionated by different centrifugation protocols in Percoll density-gradients. Staining with acridine orange under conditions denaturing RNA but leaving intact double stranded DNA yielded green fluorescence of DNA and red phosphorescence of RNA. Both light emissions were documented and quantitative image analyses were performed. By computing the integral intensities of green and red light, quantitative measures were gained for DNA and RNA contents. By correlating these light emissions to each other or to cell size, histograms characteristic for given neoblast pools were obtained. The protocol described should facilitate monitoring the heterogeneity of the neoblast compartment as well as studying cellular dynamics during growth, regeneration and other physiological processes.