Electrophoresis 1989,10, 690-697 690 Determination of the electrophoretic mobility of chromosomes by free flow electrophoresis I. Morphology and stability Isolated metaphase chromosomes of several fibroblastoid cell lines (Chinese hamster, Chinese hamster x human hybrid) were subjected to free flow electrophoresis (FFE) to study their electrophoretic mobility (EM). The morphology and stability of the chromosomes were unaffected by PFE as examined by cytogenetic methods and flow cytometry. The chromosomes of the complement all showed similar EM under most of the conditions applied. At neutral pH the EM of the chromosomes had the same sign as free DNA and about 2/3 of its magnitude. The variation of EM with bufer parameters such as ionic strength, valence of counterions, buffer capacity and dielectric constant of the solvent were investigated. Thermal denaturation increased the EM of the chromosomes by 20 %. Partial denaturation might offer a possibility to separate or enrich large amounts of chromosomes by FFE. 1 Introduction 2 Materials and methods Free flow electrophoresis (FFE), as developed by Hannig [1], is commonly used to separate cells, cell organelles or macromolecules (for reviews see [2,3]). Under the influence of electric forces, such biological particles show a characteristic electrophoretic mobility (EM) depending on their charge density and the physicochemical conditions of the buffer used. According to different EM, particles can be separated electrophoretically. The carrier free system FFE has the following characteristics: (i) the electrophoresis buffer streams vertically through a separation chamber with a typical thickness of about 0.5 mm; (ii) the homogeneous electric field is arranged perpendicularly to the flow direction of the buffer; (iii) the sample is introduced to the chamber parallel to the streaming direction and flows continuously through the electric field (zone electrophoresis). A horizontal window in the lower part of the chamber may allow observation of the sample movement with a scanner that measures the extinction at the required wavelength or light scatter intensity. Chromosomes consist of negatively charged DNA and a variety of differently charged proteins with atypical composition of about 30% DNA. 65% proteins and 5% RNA according to Nagl [4]. The protein composition of the metaphase chromosomes depends on the preparation method [5,6]. In this article the influence of FFE on the stability and morphology of chromosomes under a variety of FFE conditions was investigated. The influence of the different parameters determining EM was tested. To our knowledge this is the first time that chromosomes were successfully subjected to FFE. 2.1 Isolation of chromosomes All cell culture reagents were purchased from Serva (Heidelberg, FRG). Cells of the Chinese hamster cell line CHL and the Chinese hamster x human hybrid line ADA 13SC3 (kindly provided by P. Pearson, Leiden) were cultivated in Ham's F10 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (10000E/10000µg/mL) in a humidified CO 2 incubator (5%) at 37°C. Mitotic cells were collected overnight by a Colcemid-block (0.25µg/mL medium) and harvested by shake-off. The mitotic cells from 10 culture flasks (175 cm 2 each) were pelleted by centrifugation (350 g; 15 min), chilled for 2 min at -20 °C and transferred to 4 °C. After 10 min the chromosome pellet was suspended in a hypotonic solution (10 mM Tris, 5 mM CaCI 2 , 10 mM NaCI, pH 7.5) and incubated for 15 min at room temperature. The hypotonic solution was removed by centrifugation and the chromosome pellet resuspended in 2 mL hexandiol buffer (0.75 M 1,6- hexanediol, 25 mM Tris/HCl, pH 7.5, 5 mM MgCl 2 , 5 mM CaCI 2 , final pH adjusted to 3.2 with concentrated HAc), a modification of the hexylene glycol isolation buffer described by Stöhr and co-workers [7,8]. In the same buffer the mitotic cells were sonicated to obtain a suspension of dispersed chromosomes. The yield was between 10 8 -10 9 chromosomes. This suspension was centrifuged at 400 g for at least 10 min. All reagents were of analytical grade. 2.2 FFE of chromosomes The hexandiol buffer was removed and the Frank F. Bier 1 Ulrich Bettag 1 Thomas Rheingans 1 Hedwig Adrian 1 Jochen Barths 2 Michael Hausmann 1 Hans-Jorg Bühring 3 Peter Rohwer 4 Jürgen Dölle 1 Christoph Cremer 1 1 Institute of Applied Physics I, University of Heidelberg 2 Coultronics France SA, Margency 3 Medical Clinic II, FACS-Laboratory, University of Tübingen 4 Institute of Clinical Immunology and Rheumatology, EPICS-Laboratory, University of Erlangen Abbreviations: CHL, Chinese hamster cell line; DAPI, 4,6- diamidino-2-phenylindole; EM, electrophoretic mobility; FFE, free-flow electrophoresis; HAc, acetic acid; PI, propidium iodide