Discrimination of myogenic and nonmyogenic cells from embryonic skeletal muscle by 90 degrees light scattering

Abstract

A myogenic cell suspension was isolated from the breast muscles of 10-day-old chicken embryos by trypsin digestion. The cell preparation was subjected to Percoll density centrifugation to reduce the number of fibroblast-like cells present. The Percoll-isolated, predominantly myogenic cell population was then fractionated by flow cytometry using 90 degrees light scattering as the parameter for sorting. Cells exhibiting lower scatter, with a peak of 45 units, produced cultures containing myotubes and gave rise only to myogenic clones. Cells exhibiting higher scatter (120-200 units) produced nonmyogenic cultures and gave rise to nonmyogenic clones. Cells with intermediate light scatter were also detected. The latter produced both myogenic and nonmyogenic clones. The differences in light scatter presumably reflect higher cytoplasmic complexity of the nonmyogenic cells compared with the myogenic cells. Moreover, the differences in light scattering properties of the different cell types offer a means for the isolation of pure populations of myogenic cells directly from the intact muscle.

Fig. 1

Light scatter analysis of a representative myogenic cell fraction isolated by Percoll density centrifugation. a: A contour plot of the bivariate forward angle scatter versus 90° scatter data. b: A 90° light scatter histogram from the same experiment as in a. Regions A–D show the four regions used for sorting subpopulations. Forward and 90° scatter units are channel numbers in arbitrary units.

Fig. 2

Phase micrographs of cultures prepared from the cells in the different 90° light scatter regions. a–d: Representative fields from Figure 1, regions A–D, respectively. Cultures were initiated with 4 × 10⁴ cells per 35 mm dish and maintained for 3 days. a–c bar = 48 μm; d, 30 μm.