0196-4763/80/OlOl-0042$00.~/0 zyxwvutsrqpon CYTOMETRY Copyright zyxwvutsrqponm 0 by The Society for Analytical Cytology Vol. 1, No. 1 1980 Printed in USA. Cytometry Automated Absorption Image of Electromagnetically Exposed Frog Erythrocytes 1, zyx 2 A. Chiabrera, R. Viviani, G. Parodi, G. Vernazza, M. Hinsenkamp, A. A. Pilla, J. Ryaby, F. Beltrame, M. Grattarola and C. Nicolini SIB, Electrical Engineering Department, University of Genoa, Genoa, Italy (A.C., R.V., G.P., G.V., F.B., M.G.); Orthopaedic Surgery Department, Brugmann University Hospital, Interdisciplinary Center of Bone Biomechanics, Brussels Free University, Brussels, Belgium (M.H.); Bioelectrochemistry Laboratory, Orthopaedic Research Laboratories, College of Physicians and Surgeons, Columbia University, New York, New York (A.A.P., J.R.); Division of Biophysics, Temple University Health Sciences Center, Philadelphia, Pennsylvania (C.N.) Received for publication January 2, 1980; accepted May 2, 1980 Frog erythrocytes dedifferentiate in vitro when exposed to a suitable electromagnetic field in a chemically potentiated microenvi- ronment. These electrochemical stimuli are able to induce a chromatin decondensation, as previously shown by measuring the chro- matin accessible sites for acridine orange intercalation by means of flow cytometry (acridine orange green fluorescence). Auto- mated absorption image cytometry of Feul- gen stained smeared erythrocytes has been performed to further elucidate the above processes. After measuring or computing the area A of the nucleus, which is related to its volume, the nucleic integrated optical density D, which is related to the DNA con- tent, the average optical density DA zyxwvu = D/A, which is related to chromatin conformation, and the accessible chromatin sites SA = D213 All3, the following results have been ob- tained: zyxwvut (a) electromagnetically exposed cells progress to stages which correspond to val- ues of D equal to those of controls, in the potentiating solution, whereas A increases, zyx so that DA is smaller in exposed erythrocytes than in control ones, confirming that chro- matin decondensation is occurring as an early dedifferentiation step. (b) By using the electromagnetic signal that is most effective in promoting dedifferentiation, erythrocytes further progress toward more advanced stages, which correspond to larger values of both D and A than in controls, zyx ie., to larger DNA content. (c) In all cases, the histograms of SA are in agreement with those previously obtained by flow microfluorometry of chro- matin conformation (acridine orange green fluorescence). Finally, flow microfluorome- tric measurements of acridine orange red fluorescence give an increase of RNA con- tent for case (a), as compared with controls, and for case (b) as compared with (a). These results point out that frog erythrocytes can be electromagnetically reactivated, resum- ing both RNA and DNA syntheses after ini- tial chromatin decondensation. Frog erythrocytes are nucleated cells that undergo morpho- logic changes when exposed to suitable electromagnetic (em) fields in a chemically potentiated microenvironment zy (I, 6, 12, 14). These changes mimic those occurring in vivo within the hematoma of a frog fracture and leading to the onset of the osteogenesis process. According to Becker and Murray (l), erythrocytes dedifferentiate toward a totipotent stage, from which they differentiate dong an osteoblastic line. crofluorometry has been proposed and successfully used as a ’ Supported in part by CNR (National Research Council of Italy); National Science Foundation Grant Apr7619469; National Institutes of Health Grants CA18258, CA20034 and AM07822; and Electro- Biology Incorporated. vember 25-30, 1979. * Presented at Automated Cytology VII, Asdomar, California, No- In the Previous companion Paper zyxw (6), automated flow mi- 42